Anti atg16l1

TBT and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer, fetal bovine serum (FBS), RPMI 1640 medium, and Western Bright Enhanced Chemiluminescence (ECL) detection reagents were obtained from Thermo Fisher Scientific (Suwanee, GA, USA). An Annexin V/PI apoptosis detection kit was purchased from Multi-Sciences (Hangzhou, China). Anti-Bip (Cat#3177), anti-Calnexin (Cat#2679), anti-Ero1-Lα (Cat#3264), anti-IRE1α (Cat#3294), anti-PDI (Cat#3501), anti-CHOP (Cat#2895), anti-PERK (Cat#5683), anti-eIF2α (Cat#5324), anti-P-eIF2α (Cat#3398), anti-Atg12 (Cat#4180), anti-Beclin-1 (Cat#3495), anti-JNK (Cat#3708), anti-P-JNK (Cat#4668), anti-LC3A/B (Cat#12741), anti-Atg5 (Cat#12994), anti-Atg16L1 (Cat#8089), anti-Atg7 (Cat#8558), anti-Atg3 (Cat#3415), anti-rabbit IgG (Cat#7074), and anti-mouse IgG (Cat#7076) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-ATF6 (Cat#ab227830) was purchased from Abcam (Cambridge, UK) and anti-GAPDH was obtained from KangChen Biotech (Shanghai, China).

Equal amounts of protein were analyzed by immunoblot as previously described [14 (link), 37 (link), 39 (link)]. The transferred membranes were probed with primary antibodies against phosphorylated (p-)Akt (Cell Signaling, Beverly, MA), (p-)ERK (Santa Cruz Biotechnology, Santa Cruz, CA) or (p-)JNK (New England BioLabs, Beverly, MA), or anti-ATG16L1, LC3B (Cell Signaling, Beverly, MA) or NOD2 (Cayman Chemical, Ann Arbor, MI). After washes, the membranes were incubated with appropriate horseradish peroxidase-associated secondary antibodies before signals were visualized with the enhanced chemiluminescence detection system (Amersham Bioscience).

Cells were fixed in 2% freshly depolymerized paraformaldehyde and 0.2% glutaraldehyde in 0.1 M cacodilate buffer pH 7.4 for 1 h at 4 °C. Samples were rinsed in the same buffer, partially dehydrated and embedded in London Resin White (LR White). Ultrathin sections were processed for immunogold technique. Grids were pre-incubated with 10% normal goat serum in 10 mM PBS containing 1% bovine serum albumine (BSA) and 0.13% NaN₃ (medium A), for 15 minutes at room temperature. Sections were then incubated with primary antibody, mouse anti-RTN-1C (Abcam, ab8961) diluted 1:100 in medium A for 1 h at room temperature. After rinsing in medium A containing 0.01% Tween 20 (Merck), sections were incubated in goat anti-mouse IgG conjugated to 15 nm colloidal gold (British BioCell Int. EM.GMHL15), diluted 1:30 in medium A, containing fish gelatine, for 1 h at room temperature. The double immunolabeling has been performed incubating the RTN-1C labeled sections with rabbit polyclonal anti-ATG16L1 (Cell Signaling, 8089) followed by goat anti-rabbit IgG conjugated to 5 nm colloidal gold (British BioCell Int. EM.GAR5), diluted 1:30 in medium A, containing fish gelatine, for 1 h at room temperature. Grids were thoroughly rinsed in distilled water, contrasted with aqueous 2% uranyl acetate for 20 min. and photographed in a Zeiss EM 900 electron microscope.

Antibodies used in these experiments included the following: anti‐cleaved‐caspase3 (ab32042, Abcam), anti‐caspase3 (NB100‐56708SS, Novus), anti‐caspase9(ab32539, Abcam), anti‐BAX (50599‐2‐Ig, Proteintech), anti‐Bcl‐2 (GTX100064, GeneTex) anti‐GAPDH (#5174, Cell Signaling Technology), anti‐P62 (M162‐3, Medical Biological Laboratories), anti‐Beclin1 (11306‐1‐AP, Proteintech), anti‐LC3B (GB11124, Servicebio), anti‐ATG16L1(#8089, Cell Signaling Technology), anti‐ATG7(#8558, Cell Signaling Technology), anti‐p53(SC‐126, Santa Cruze), and anti‐ubiquitin(10201‐2‐AP, Proteintech).
Reagents used in these experiments included the following: autophagy inhibitor 3‐Methyladenine (3‐MA) (S2767, Selleck), p53 pathway inhibitor Pifithrin‐α (PFT‐α) (S2929, Selleck), temozolomide (TMZ) (S1237, Selleck), MG132 (S2619, Selleck), and cycloheximide (CHX)(HY‐12320, MCE).

Cells grown in 6‐well format were washed in ice‐cold PBS and scraped in 100 μl of Lysis Buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton X‐100, 5 mM EDTA and protease inhibitors) at the relevant time. Protein concentration was estimated using 660 nm Protein Assay Reagent (Pierce) and 20–30 μg of protein loaded on SDS–PAGE gel. Antibodies used were anti‐TECPR1 (8097, Cell Signalling), anti‐ATG5 (A0731, Sigma), anti‐ATG16L1 (8089, Cell Signalling), anti‐GFP (JL8, Clontech) and anti‐β Actin (ab8227, Abcam).