Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: ATG9A knockout HAP1 cell lysate (20 µg)
Lane 3: HepG2 cell lysate (20 µg)
Lane 4: A375 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab108338 observed at 100 and 130 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab108338 was shown to specifically react with ATG9A when ATG9A knockout samples were used. Wild-type and ATG9A knockout samples were subjected to SDS-PAGE. ab108338 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 and incubated overnight at 4°C. 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 1/10,000 dilution for 1 h at room temperature before imaging.

Accumulation of ATG9A at the TGN of AP-4 μ4 mutant patient fibroblasts.

Skin fibroblasts were from one control individual and two patients homozygous for mutations in the AP4M1 gene encoding AP-4 μ4.

Co-immunostaining for endogenous ATG9A (green) and AP-4 ε (red) (B) or GM130 of the fibroblasts.

ATG9A is detected using ab108338.

 

(From Figure 4B of De Pace et al)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human thyroid carcinoma tissue sections labeling ATG9A with Purified ab108338 at 1:50 dilution (4.12 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with purified ab108338 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 100% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ATG9A with Purified ab108338 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/10000 dilution (purified)

Lane 1 : 293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in non-boiled method
Lane 2 : 293 (Human embryonic kidney epithelial cell) whole cell lysate prepared in boiled method
Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in non-boiled method
Lane 4 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared in boiled method

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

We suggest not to boil the sample after lysis.
Blocking and diluting buffer: 5% NFDM/TBST

Exposure time:  
Left image: 5 seconds
Right image: 2 seconds

 

ab108338 (purified) at 1:20 dilution (2μg) immunoprecipitating ATG9A in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate.

Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2 (+): ab108338 & HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108338 in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution. No band in input lane is due to the boiled lysates
Blocking and diluting buffer: 5% NFDM/TBST.

Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution (purified) + Mouse spinal cord lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa

Blocking and diluting buffer: 5% NFDM/TBST. 

The lysates are boiled.

Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/2000 dilution (purified) + Rat brain lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

The lysates are boiled.

Unpurified ab108338 staining ATG9A in the human cell line HepG2 (human hepatocellular carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/40. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

All lanes : Anti-ATG9A antibody [EPR2450(2)] (ab108338) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : 293T cell lysate
Lane 3 : A375 cell lysate
Lane 4 : Mouse brain cell lysate
Lane 5 : Rat brain cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 94 kDa

Unpurified ab108338, at 1/100, staining ATG9A in paraffin-embedded Human colon tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Unpurified ab108338 at 1/50 dilution, staining ATG9A in HepG2 (Human hepatocellular carcinoma epithelial cell) cells by Immunofluorescence.