IHC images of vessel staining of ab195242, ATG16L1 (phospho S278), in sections of formalin-fixed paraffin-embedded normal human skeletal muscle tissue*, performed on a Leica BOND^TM system using a modified protocol F.

Both sections were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. One section was then treated with 200 enzyme units of alkaline phosphatase (AP+) for 1 hour at 37°C; and the other in buffer containing no alkaline phosphatase (AP-) for 1 hour at 37°C. The sections were then incubated with ab195242, 3µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The sections were then counterstained with haematoxylin and mounted with DPX.

Identical assays were also performed using detection system-only (no primary antibody) as reagent controls (data not shown), to ensure that staining seen was a result of the binding of the primary antibody.

The absence of staining in the AP+ tissue compared to the AP- tissue adds further evidence of phospho specificity for this antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC images of mice quadricep showing either pATG16L1 or LC3B staining

Mice were fed ad libitum or starved for 16 hours. Quadricep muscle were immediately harvested and fixed in 10% formalin for 2 days. The samples were then paraffin embedded, sectioned into 4µm thick slices, and mounted onto glass microscope slides.  Slides were stained with primary antibody overnight at 4˚C: LC3B 1/1000, pATG16L1 (ab195242) 1/300. Secondary antibody: Alexa Fluor 555 anti-rabbit, 1/1000.

IF showing pATG16L1 (red) and total ATG16L1 (green):

Polyclonal population of ATG16L1 KO and HA-ATG16L1 reconstituted cells were starved of amino acid for 1 hour and stained. Blocking buffer used for pATG16L1 staining: 0.1% BSA, 1x abcam blocking buffer ab126587, diluted in PBS. Anti-pATG16L1 (ab195242) concentration: 1/150. Anti-ATG16L1, concentration: 1/200 Secondary antibody (Alexa Fluor 647/488) concentration: 1/1000.

IF showing pATG16L1 (red), LC3B (green) and p62 (white):

MEF cells were amino acid starved for 1 hour. Blocking buffer used for pATG16L1 (ab195242): 0.1% BSA, 1x abcam blocking buffer (ab126587), diluted in PBS. Anti-pATG16L1 (ab195242) concentration: 1/150. Secondary antibody (Alexa Fluor 647) concentration: 1/1000

HCT116 wild-type and ATG16L1 knockout cells were incubated with either complete media or amino acid deficient DMEM for 3 hours. 5ug of whole cell lysate were resolved by SDS-PAGE on a 6%-18% gradient gel, then transferred onto PVDF membrane. Membrane was blocked in 10X blocking buffer (Cat # ab126587) diluted in TBS solution for 30 minutes; incubated with 1:1000 primary antibody in 2.5% BSA TBST solution overnight at 4°C ; incubated with 1:15000 secondary antibody in 2% milk TBST solution for 45 minutes. Immobilon ECL was applied for 1 minute then imaged with film.

All lanes : Anti-ATG16L1 (phospho S278) antibody [EPR19016] (ab195242) at 1/1000 dilution

Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 (WT) expression vector containing a myc-His-tag®, followed by treatment with alkaline phosphatase for 1 hour, whole cell lysate
Lane 4 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 S278A expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 68 kDa
Observed band size: 68 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Dot blot analysis of ATG16L1 (phospho S278) labeled with ab195242 at 1/1,000 dilution.

Lane 1: Mouse ATG16L1 (phospho S278) peptide;

Lane 2: Mouse ATG16L1 non-phospho peptide;

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100,000 dilution was used as secondary antibody.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.