Anti-p18 INK4c/CDKN2C antibody [EPR15891]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Immunofluorescent analysis of HeLa cells (4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized) labeling p18 INK4C/CDKN2C with ab192239 at 1/100 dilution (5μg/mL) followed by Goat anti rabbit IgG (AlexaFluor® 488) (ab150077) secondary at 1/200 dilution and counter-stained with DAPI (blue).
Negative controls : anti-p18 INK4C/CDKN2C at 1/100 dilution, Secondary ab (Goat anti mouse IgG (Alexa Fluor®594)) at 1/400 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Immunohistochemical analysis of paraffin-embedded Human glioma tissue labeling p18 INK4C/CDKN2C with ab192239 at 1/50 dilution followed by pre-diluted HRP Polymer for Rabbit/Mouse IgG secondary antibody and counter-stained with Hematoxylin. (inset : negative control).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Immunohistochemical analysis of paraffin-embedded Human brain tissue labeling p18 INK4C/CDKN2C with ab192239 at 1/50 dilution followed by pre-diluted HRP Polymer for Rabbit/Mouse IgG secondary antibody and counter-stained with Hematoxylin. (inset : negative control).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Western blot analysis of immunoprecipitation pellet from HeLa lysate immunoprecipitated using ab192239 at 1/30 dilution.
Secondary : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution.

All lanes:

Immunoprecipitation - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (ab192239)

Predicted band size: 18 kDa

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• WB

Lab

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Lanes 1-4 : Merged signal (red and green). Green - ab192239 observed at 18 kDa. Red - loading control ab8245 observed at 37 kDa.

ab192239 Anti-p18 INK4c/CDKN2C antibody [EPR15891] was shown to specifically react with Cyclin Dependent Kinase Inhibitor 2C in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265031 (knockout cell lysate ab257887) was used. Wild-type and Cyclin Dependent Kinase Inhibitor 2C knockout samples were subjected to SDS-PAGE. ab192239 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (ab192239) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CDKN2C knockout HeLa cell lysate at 20 µg

Lane 3:

Rat kidney cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 18 kDa

false

• WB

Supplier Data

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

All lanes:

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (ab192239) at 1/10000 dilution

Lane 1:

293 cell lysate at 20 µg

Lane 2:

Ramos cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 18 kDa

false

• WB

Supplier Data

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

All lanes:

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (ab192239) at 1/1000 dilution

Lane 1:

Rat kidney lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Lane 3:

NIH 3T3 lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 18 kDa

false

• WB

CiteAb

Western blot - Anti-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Western Blotting using Anti-p18 INK4c/CDKN2C antibody [EPR15891], ab192239. 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-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Western Blotting using Anti-p18 INK4c/CDKN2C antibody [EPR15891], ab192239. 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-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Western Blotting using Anti-p18 INK4c/CDKN2C antibody [EPR15891], ab192239. 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-p18 INK4c/CDKN2C antibody [EPR15891] (AB192239)

Western Blotting using Anti-p18 INK4c/CDKN2C antibody [EPR15891], ab192239. 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