Anti-YTHDF2 antibody [EPR20318]

• IP

Lab

Immunoprecipitation - Anti-YTHDF2 antibody [EPR20318] (AB220163)

ab220163 Immunoprecipitating YTHDF2 in human HepG2 whole cell lysate. 10 μg of cell lysate was incubated with primary antibody (1/30). For western blotting ab220163 (1/1000) was used to confirm successful immunoprecipitation. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at (1/5000). For lane 3 Rabbit monoclonal IgG (ab172730) was used instead of ab220163.

In panel A, the lysate was stored at -80^oC prior to the IP and incubation with the primary antibody was carried out overnight. In panel B, lysate was freshly made and used in the IP test immediately to minimize protein degradation. The incubation time was also shortened from overnight to 2h.

All lanes:

Immunoprecipitation - Anti-YTHDF2 antibody [EPR20318] (ab220163)

Predicted band size: 62 kDa

Observed band size: 62 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

Blocking/diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/5000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 10μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 4 hours, whole cell lysate at 10 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: 62 kDa

false

Exposure time: 103s

• WB

Lab

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

Blocking/diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/5000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 10μM MG-132 (<a href='/en-us/products/biochemicals/mg-132-proteasome-inhibitor-ab141003'>ab141003</a>) for 4 hours, whole cell lysate at 10 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

false

Exposure time: 103s

• WB

Supplier Data

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST

The wild-type and YTHDF2 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220163 was shown to specifically react with YTHDF2 in wild-type mESC cells as signal was lost in YTHDF2 knockout cells. Wild-type and YTHDF2 knockout samples were subjected to SDS-PAGE. ab220163 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.

All lanes:

Western blot - Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1:

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

Lane 2:

YTHDF2 knockout mESC whole cell lysate at 20 µg

Lane 3:

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: 62 kDa

false

Exposure time: 48s

• WB

Unknown

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

Blocking/diluting buffer and concentration : 5% NFDM/TBST

Exposure time : Lane 1 : 3 minutes; Lane 2 : 92 seconds; Lane 3 : 37 seconds; Lane 4 : 8 seconds; Lane 5 : 15 seconds.

All lanes:

Western blot - Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 10 µg

Lane 2:

Mouse testis tissue lysate at 10 µg

Lane 3:

Rat testis tissue lysate at 10 µg

Lane 4:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 10 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lanes 2 - 5:

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: 62 kDa

false

• WB

Supplier Data

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

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

All lanes:

Western blot - Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1:

GST-tagged human YTHDF1 recombinant protein, 20ng

Lane 2:

GST-tagged human YTHDF2 recombinant protein, 20ng

Lane 3:

GST-tagged human YTHDF3 recombinant protein, 20ng

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: 62 kDa

false

Exposure time: 3s

• WB

CiteAb

Western blot - Anti-YTHDF2 antibody [EPR20318] (AB220163)

YTHDF2 western blot using anti-YTHDF2 antibody [EPR20318] ab220163. Publication image and figure legend from Jin, D., Guo, J., et al., 2020, Mol Cancer, PubMed 32106857.

ab220163 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 ab220163 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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