Atg9a

We used the antibodies in parentheses to the following antigens: ATG9A (Abcam, catalog #108338), FLAG M2 (Sigma, F1804), PAK3 (Abnova, PAB2300), SPTLC2 (Abcam, ab23696), GFP-HRP (MACS, 130091833), actin-HRP (Sigma, A3854), beclin 1 (Cell Signaling, 3738), ATG7 (Cell Signaling, 8558), ATG5 (Cell Signaling, 12994), ATG12 (Cell Signaling, 4180), ATG16 (Cell Signaling, 8089), LC3 (Cell Signaling, 3868), LAMP-1 (Cell Signaling, 9091), SNAP29 (Abcam, 138500), gp41 (NIH AIDS Reagent Program, 2F5), TGN46 (Bio-Rad, AHP500G), anti-HIV immunoglobulin (NIH AIDS Reagent Program, HIV-Ig), Nef (NIH AIDS Reagent Program, 2949), α-tubulin (Sigma, T5168), VSV-G (Sigma, V5507), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen, A21206), Alexa Fluor 488- conjugated donkey anti-mouse IgG (Invitrogen, A21202), Alexa Fluor 555-conjugated donkey antirabbit IgG (Invitrogen, A31572), Alexa Fluor 555-conjugated donkey anti-mouse IgG (Invitrogen, A31570), Alexa Fluor 555-conjugated donkey anti-sheep IgG (Invitrogen, A21436), HRP-conjugated donkey anti-rabbit IgG (GE Healthcare, NA934V), and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, NXA931).

Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from Gibco. Earle's balanced salt solution (EBSS) and propranolol were purchased from Sigma‐Aldrich. Rapamycin (RAPA), 3‐methyladenine (3‐MA) and chloroquine (CQ) were obtained from MedChemExpress, and ZVAD‐FMK, necrostatin‐1, liproxstatin‐1, SB203580, SP600125, SC79 and 740Y‐P were purchased from SelleckChem. Then, these reagents were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals or water and stored at −80°C. Primary antibodies against Akt, p‐Akt, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, Erk1/2 and p‐Erk1/2 were purchased from Cell Signalling Technology. Primary antibodies against alpha‐smooth muscle actin (α‐SMA), fibronectin (FN), LC3B, P62, p‐PI3K p85, PI3K p85, ATG9b, ATG9a, mTOR, p‐mTOR, ATG12, ATG5 and anti‐ubiquitin were procured from Abcam. Primary antibodies against beta‐actin, alpha‐tubulin, GAPDH and HRP‐conjugated secondary antibodies were obtained from Proteintech. DyLight 549‐conjugated and DyLight 488‐conjugated secondary antibodies were provided by Abbkine.

Cells were infected with GAS as described above. At indicated times post infection, cells were lysed with RIPA lysis buffer containing protease inhibitor (Promega). Cell lysates were quantified using Bradford protein assay (Bio-Rad, Hercules, CA, United States) and boiled in SDS sample buffer for 10 min. All samples were then separated by 12% SDS-polyacrylamide gel and transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA, United States). After blocking with 5% skim milk, the membranes were incubated with primary antibody against ATG9A (Abcam Technology, Cambridge, MA, United States), LC3 (MBL, Nagoya, Japan), or β-actin (novusbio, Littleton, CO) in blocking buffer at 4°C for overnight. After incubation, horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, United States) were used to visualize the Western blot. All images were acquired with the ImageQuant LAS-4000 imaging system (GE Healthcare Life Sciences, Pittsburgh, PA, United States).