Anti-YTHDF1 antibody [EPR22349-41]

• IP

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Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

YTHDF1 was immunoprecipitated from 0.35 mg HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate with ab220162 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220162 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.
Lane 1 : HepG2 whole cell lysate 10 μg (Input).
Lane 2 : ab220162 IP in HepG2 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220162 in HepG2 whole cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

All lanes:

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (ab220162)

Predicted band size: 61 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

YTHDF1 was immunoprecipitated from HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab220162 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220162 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : HeLa whole cell lysate 10 μg (Input). Lane 2 : ab220162 IP in HeLa whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220162 in HeLa whole cell lysate. Blocking/Dilution buffer : 5% NFDM/TBST. Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (ab220162) at 1/1000 dilution

All lanes:

HeLa whole cell lysate at 10 µg

Predicted band size: 61 kDa

Observed band size: 60 kDa

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Exposure time: 8s

• WB

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Western blot - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

The wild-type and YTHDF1 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220162 was shown to specifically react with YTHDF1 in wild-type mESC cells as signal was lost in YTHDF1 knockout cells. Wild-type and YTHDF1 knockout samples were subjected to SDS-PAGE. ab220162 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Exposure times : Lanes 1 and 2 : 59 seconds; Lanes 3 and 4 : 26 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (ab220162) at 1/1000 dilution

Lane 1:

Wild-type mESC (mouse embryo stem cell) whole cell lysate at 20 µg

Lane 2:

YTHDF1 knockout mESC whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

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• WB

Supplier Data

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Blocking and diluting buffer and concntration : 5% NFDM/TBST Imaging carried out using iBright CL 1000 Imaging System

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (ab220162) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate 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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 60 kDa

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Exposure time: 180s

• WB

Unknown

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

The YTHDF recombinant proteins were kindly provided by an anonymous collaborator.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (ab220162) at 1/1000 dilution

Lane 1:

GST-tagged human YTHDF1 recombinant protein, 20 ng

Lane 2:

GST-tagged human YTHDF2 recombinant protein, 20 ng

Lane 3:

GST-tagged human YTHDF3 recombinant protein, 20 ng

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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 87 kDa

false

Exposure time: 3s

• WB

CiteAb

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (AB220162)

YTHDF1 western blot using anti-YTHDF1 antibody [EPR22349-41] ab220162. Publication image and figure legend from Jin, D., Guo, J., et al., 2020, Mol Cancer, PubMed 32106857.

ab220162 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab220162 please see the product overview.

YTHDF1 and YTHDF2 competitively interacted with YTHDF3 in an m6A- independent manner to regulate YAP expression. (a) The diagram of that YTHDF1 and YTHDF2 competitively interacted with YTHDF3 to regulate YAP expression in NSCLC. (b, c) The interaction between YTHDF1/2/3 and YAP mRNA was determined by RIP assay. (d) The interactions between YTHDF1/YTHDF2 and the mRNA of YAP were increased when YTHDF3 is existed determined by RNA pulldown assay. (e) Western blot analysis indicated that FTO and ALKBH5 were only in nuclear fractions but YTHDF1/2/3 were only in cytoplasm fractions in A549 cells. (f, g) Immunofluorescent staining showed that ALKBH5 WT/KD were only in nuclear fractions (f) but YTHDF1/2/3 were only in cytoplasm fractions (g) in A549 cells. (h) Co-IPs performed using lysates collected from A549 cells with immunoprecipitation by either YTHDF1, YTHDF2 or YTHDF3 antibodies. (i) The protein level of YTHDF3 was analyzed in lysates collected from either YTHDF1 or YTHDF2 transfected A549 and H1299 cells determined by Co-IPs assays by immunoprecipitation with YTHDF2 or YTHDF1 antibodies, respectively. (j, k) The interactions between YTHDF1/2/3 and YAP mRNA were detected in A549 and H1299 cells with transfection with indicated genes determined by Co-IP assay using the MS2 coat protein system. (l) The interactions between YTHDF1/2 and YAP mRNA was determined by RIP assay in A549 and H1299 cells with transfection of indicated genes. (m) The m6A levels of YAP were analyzed by in A549 and H1299 cells with transfection into indicated genes. (n) The protein level of YTHDF3 was detected in lysates collected from A549 cells determined by Co-IP assay with immunoprecipitation with either YTHDF1 or YTHDF2 antibodies. Results were presented as mean ± SD of three independent experiments. *p < 0.05 or **p < 0.01 indicates a significant difference between the indicated groups. ns, not significant

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