All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution

Lane 1 : Wild-type mESC (mouse embryo stem cell) whole cell lysate
Lane 2 : YTHDF3 knockout mESC whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 64 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?


Exposure time: 59 seconds

The wild-type and YTHDF3 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220161 was shown to specifically react with YTHDF3 in wild-type mESC cells as signal was lost in YTHDF3 knockout cells. Wild-type and YTHDF3 knockout samples were subjected to SDS-PAGE. ab220161 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.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling YTHDF3 with ab220161 at 1/100 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human colon (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in mouse cerebrum (PMID:29103884, 28250115) is observed. Counterstained with DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling YTHDF3 with ab220161 at 1/60 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

YTHDF3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab220161 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220161 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg (Input).
Lane 2: ab220161 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab220161 in HeLa whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 15 seconds.

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

All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution

Lane 1 : HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate (fresh lysate)
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate (fresh lysate)
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (fresh lysate)
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate (fresh lysate)
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 64 kDa

Lysate should be made freshly and used in WB immediately to minimize protein degradation (lane1-4). Lane 5-7 are the lysates from same cell lines but have experienced freeze-thaw cycles.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-4: 37 secs; Lanes 5-7: 92 secs.

All lanes : Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/5000 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 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 64 kDa


Exposure time: 10 seconds

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in rat cerebrum (PMID:29103884, 28250115) is observed. Counterstained with DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of rat cerebrum (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of mouse cerebrum (PMID:29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).