Anti-p63 antibody [EPR5701]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p63 antibody [EPR5701] (AB124762)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast tissue sections labeling p63 with purified ab124762 at 1/5000 dilution (0.16 μg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC

Lab

Immunohistochemistry - Anti-p63 antibody [EPR5701] (AB124762)

Immunohistochemical analysis of formalin fixed paraffin embedded human prostate labelling p63 with ab124762 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab124762 anti-p63 [EPR5701] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-p63 antibody [EPR5701] (AB124762)

Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling p63 with Purified ab124762 at 1/80 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-p63 antibody [EPR5701] (AB124762)

Immunocytochemistry/ Immunofluorescence analysis of A431(Human epidermoid carcinoma epithelial cell) cells labeling p63 with Purified ab124762 at 1/200 dilution (4 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

AbReview39942****

Immunocytochemistry/ Immunofluorescence - Anti-p63 antibody [EPR5701] (AB124762)

Unpurified ab124762 staining p63 in Human corneal limbal epithelial cells (primary culture) by ICC/IF (immunocytochemistry/immunofluorescence). Cells were fixed with methanol and permeabilized with 0.3% Triton X-100 for 5 minutes. Samples were incubated with primary antibody (1/100 in PBS + 10% Goat serum) for 18 hours at 4°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Manuel Chacon

• mIHC

Lab

Multiplex immunohistochemistry - Anti-p63 antibody [EPR5701] (AB124762)

Fluorescence multiplex immunohistochemical analysis of human prostate gland tissue (formalin/PFA-fixed paraffin-embedded section). Panel A : merged staining of anti-p63 (ab124762, magenta; Opal™690), anti-Cytokeratin 5 (ab236216, green; Opal™520) and anti-Prostate Specific Antigen (ab76113, red; Opal™570) on human prostate gland tissue. Panel B : anti-Prostate Specific Antigen stained on luminal cells. Panel C : anti-Cytokeratin 5 stained on cytoplasm of basal cells. Panel D : anti-p63 stained on nucleus of basal cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining : in the order of ab124762 (1/5000), ab236216 (1/400), and ab76113 (1/2000) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

• WB

Unknown

Western blot - Anti-p63 antibody [EPR5701] (AB124762)

Blocking/Diluting buffer : 5% NFDM/TBST

The bands observed are consistent with what have been described in PMID 30649915 as isoforms of p63.

All lanes:

Western blot - Anti-p63 antibody [EPR5701] (ab124762) at 1/1000 dilution

Lane 1:

HaCaT (Human skin keratinocyte) whole cell lysates at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

Mouse skin lysates at 20 µg

Lane 4:

Mouse thymus lysates at 20 µg

Lane 5:

Mouse bladder lysates at 20 µg

Lane 6:

Rat skin lysates at 20 µg

Lane 7:

Rat bladder lysates at 20 µg

Lane 8:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 77 kDa

Observed band size: 37-75 kDa

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• WB

CiteAb

Western blot - Anti-p63 antibody [EPR5701] (AB124762)

Western Blotting using Anti-p63 antibody [EPR5701], ab124762. Publication image from Liu, M. et al., 2020, Theranostics, 32292506. Legend direct from paper.

Identification of downstream targets of PRMT5 in gastric cancer cells. (A) Relative mRNA expression levels of a panel of key genes involved in cell proliferation and cell cycle regulation were analyzed by quantitative real-time PCR in scrambled control (Scr) or PRMT5-silenced (PRMT5 sh1/2) BGC823 and SGC7901 cells. GAPDH was used as an endogenous control. Data shown are mean ± SD (n = 3). **P < 0.01. (B) Immunoblots of PTEN, p18, p21, p57 and p63 protein levels in PRMT5-silenced BGC823 and SGC7901 cells. GAPDH was served as a loading control. (C) PTEN, p18, p21, p57 and p63 knockdown by shRNAs were verified by Western blot in PRMT5-silenced BGC823 cells. HSP70 was served as a loading control. (D) Cell proliferation was assessed by CCK-8 assay at days 1, 2, 3 and 4 in Scr, sh-PT5-treated, sh-PT5 + sh-PTEN-treated, sh-PT5 + sh-p18-treated, sh-PT5 + sh-p21-treated, sh-PT5 + sh-p57-treated or sh-PT5 + sh-p63-treated BGC823 cells. Data shown are mean ± SD (n = 3). **P < 0.01. (E) Colony-formation was determined in Scr, sh-PT5-treated, sh-PT5 + sh-PTEN-treated, sh-PT5 + sh-p18-treated, sh-PT5 + sh-p21-treated, sh-PT5 + sh-p57-treated or sh-PT5 + sh-p63-treated BGC823 cells. Colony numbers are shown in the bar graph (right panel). Data shown are mean ± SD (n = 3). *P < 0.05. (F) Representative images of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenograft tumors at day 20. (G) Growth curves of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenograft tumors. Data shown are mean ± SD (n = 6). **P < 0.01. (H) Average tumor weights of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenografts (n = 6). **P < 0.01. (I) Representative images of H&E and IHC staining for PRMT5, p57 and Ki-67 expression from Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenografts. Scale bar : 20 µm. (J) Cell proliferation was assessed by CCK-8 assay at days 1, 2, 3 and 4 in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. Data shown are mean ± SD (n = 3). **P < 0.01. (K) Colony-formation was assessed in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. Colony numbers were shown in the bar graph (right panel). Data shown are mean ± SD (n = 3). *P < 0.05. (L) Immunoblots of PTEN, p18, p21, p57 and p63 protein in Scr, sh-PT5-treated, sh-PT5 + PRMT5 (wild type)-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. GAPDH served as a loading control.

false

• WB

CiteAb

Western blot - Anti-p63 antibody [EPR5701] (AB124762)

Western Blotting using Anti-p63 antibody [EPR5701], ab124762. Publication image from Liu, M. et al., 2020, Theranostics, 32292506. Legend direct from paper.

Identification of downstream targets of PRMT5 in gastric cancer cells. (A) Relative mRNA expression levels of a panel of key genes involved in cell proliferation and cell cycle regulation were analyzed by quantitative real-time PCR in scrambled control (Scr) or PRMT5-silenced (PRMT5 sh1/2) BGC823 and SGC7901 cells. GAPDH was used as an endogenous control. Data shown are mean ± SD (n = 3). **P < 0.01. (B) Immunoblots of PTEN, p18, p21, p57 and p63 protein levels in PRMT5-silenced BGC823 and SGC7901 cells. GAPDH was served as a loading control. (C) PTEN, p18, p21, p57 and p63 knockdown by shRNAs were verified by Western blot in PRMT5-silenced BGC823 cells. HSP70 was served as a loading control. (D) Cell proliferation was assessed by CCK-8 assay at days 1, 2, 3 and 4 in Scr, sh-PT5-treated, sh-PT5 + sh-PTEN-treated, sh-PT5 + sh-p18-treated, sh-PT5 + sh-p21-treated, sh-PT5 + sh-p57-treated or sh-PT5 + sh-p63-treated BGC823 cells. Data shown are mean ± SD (n = 3). **P < 0.01. (E) Colony-formation was determined in Scr, sh-PT5-treated, sh-PT5 + sh-PTEN-treated, sh-PT5 + sh-p18-treated, sh-PT5 + sh-p21-treated, sh-PT5 + sh-p57-treated or sh-PT5 + sh-p63-treated BGC823 cells. Colony numbers are shown in the bar graph (right panel). Data shown are mean ± SD (n = 3). *P < 0.05. (F) Representative images of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenograft tumors at day 20. (G) Growth curves of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenograft tumors. Data shown are mean ± SD (n = 6). **P < 0.01. (H) Average tumor weights of Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenografts (n = 6). **P < 0.01. (I) Representative images of H&E and IHC staining for PRMT5, p57 and Ki-67 expression from Scr, sh-PT5-treated or sh-PT5 + sh-p57-treated BGC823 xenografts. Scale bar : 20 µm. (J) Cell proliferation was assessed by CCK-8 assay at days 1, 2, 3 and 4 in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. Data shown are mean ± SD (n = 3). **P < 0.01. (K) Colony-formation was assessed in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. Colony numbers were shown in the bar graph (right panel). Data shown are mean ± SD (n = 3). *P < 0.05. (L) Immunoblots of PTEN, p18, p21, p57 and p63 protein in Scr, sh-PT5-treated, sh-PT5 + PRMT5 (wild type)-treated or sh-PT5 + PRMT5δ (enzymatically inactive)-treated BGC823 cells. GAPDH served as a loading control.

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• WB

CiteAb

Western blot - Anti-p63 antibody [EPR5701] (AB124762)

Western Blotting using Anti-p63 antibody [EPR5701], ab124762. Publication image from Liu, M. et al., 2020, Theranostics, 32292506. Legend direct from paper.

PRMT5-dependent direct interaction with c-Myc represses gene expression of PTEN and p57. (A) The subcellular location of c-Myc and PRMT5 proteins was documented in BGC823 and SGC7901 cells by immunofluorescence microscopy. Scale bar : 10 µm. (B) Co-immunoprecipitation of endogenous c-Myc with Flag-PRMT5 from SGC7901 cells overexpressing Flag-tagged PRMT5. IgG was used as the negative control. (C) Western blot analysis of c-Myc binding to purified GST or GST-PRMT5 fusion protein using c-Myc antibody (top). GST or GST-PRMT5 fusion protein purified from E. coli was visualized by Coomassie blue staining (bottom). An asterisk denotes the GST-PRMT5 fusion protein. (D) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5 fragments F1 (amino acids 1-354), F2 (amino acids 355-453) or F3 (amino acids 454-637) using c-Myc antibody (top). GST or GST-PRMT5 F1, F2, or F3 fusion proteins from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST-PRMT5 F1, F2, and F3 fusion proteins. (E) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5 or GST-PRMT5 488-494δ (deletion of amino acids 488-494) using c-Myc antibody (top). GST, GST-PRMT5 or GST-PRMT5 488-494δ purified from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST-PRMT5 or GST-PRMT5 488-494δ fusion proteins. (F) Western blot analysis of c-Myc binding to purified GST, GST-PRMT5, GST-PRMT5 R488A or GST-PRMT5 K490A using c-Myc antibody (top). GST, GST-PRMT5, GST-PRMT5 R488A or GST-PRMT5 K490A purified from E. coli was visualized by Coomassie blue staining (bottom). Asterisks denote the GST- PRMT, GST-PRMT5 R488A or GST-PRMT5 K490A fusion proteins. (G) Western blot analysis of H4R3me2s levels in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. Histone H4 served as a loading control. (H) Relative mRNA levels of PRMT5, PTEN, p57, p18, p21 and p63 was examined by quantitative real-time PCR assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. GAPDH was used as an endogenous control. *P < 0.05, **P < 0.01. (I) Protein levels of PRMT5, PTEN, p57, p18, p21 and p63 were examined by Western blot assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. GAPDH served as a loading control. (J) Relative enrichment of H4R3me2s at the promoters of PTEN (P4, left panel) and p57 (P5, right panel) was examined by ChIP assays in Scr, sh-PT5-treated, sh-PT5 + PRMT5 WT-treated or sh-PT5 + PRMT5 K490A-treated BGC823 cells. IgG was used as a negative control. Data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01. (K) Relative enrichment of H4R3me2s at the promoters of PTEN (P4, left panel) and p57 (P5, right panel) was examined by ChIP assays in NC, c-Myc siR-1 and c-Myc siR-2-treated BGC823 cells. IgG was used as a negative control. Data shown are mean ± SD (n = 3). **P < 0.01.

false

• WB

CiteAb

Western blot - Anti-p63 antibody [EPR5701] (AB124762)

Western Blotting using Anti-p63 antibody [EPR5701], ab124762. Publication image from Liu, M. et al., 2020, Theranostics, 32292506. Legend direct from paper.

c-Myc is co-enriched with H4R3me2s at PRMT5-targeted genes and represses their expression. (A) c-Myc is enriched at the promoters of PTEN (P4), p18 (P2), p21 (P2), p57 (P5) and p63 (P2) in BGC823 and SGC7901 cells by ChIP analysis. IgG was used as a negative control. Data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01. (B) Relative mRNA expression levels of PTEN, p18, p21, p57 and p63 were analyzed by quantitative real-time PCR in negative control (NC) or c-Myc-silenced (c-Myc siR-1/2) BGC823 and SGC7901 cells. GAPDH was used as an endogenous control. Data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01. (C) Immunoblots of PTEN, p18, p21, p57 and p63 protein levels in negative control (NC) or c-Myc-silenced (c-Myc siR-1/2) BGC823 and SGC7901 cells. GAPDH served as a loading control. (D) Representative images of IHC staining of c-Myc in adjacent noncancerous (Normal) or gastric cancer (Tumor) tissues. The boxed areas in the left images are magnified in the right images. Scale bar : 50 µm. (E) IHC score of c-Myc in gastric cancer (n = 70) and adjacent noncancerous tissues (n = 70), P < 0.05.

false