Rabbit anti atg16l1

Rabbit anti-LC3B (#3868), rabbit anti-ATG5 (#12994), rabbit anti-ATG7 (#8558), rabbit anti-Beclin-1 (#3495), rabbit anti-ATG16L1 (#8089), rabbit anti-BIP (#3177), rabbit anti-ATF6 (#65880), rabbit anti-ATF4(#11815), rabbit anti-XBP1 (#12782), rabbit anti-PERK (#5683), rabbit anti-IRE1 (#3294), rabbit anti-p-eif2α (#3398), rabbit anti-FIP200 (#12436), and mouse anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (dilution concentration 1:1000). Rabbit anti-p-IRE1 (PA1-16927) was purchased from Invitrogen (Waltham, MA, USA) (dilution concentration 1:1000). Mouse anti-p62 (18420-1-AP), mouse anti-HA-tag (66006-2-Ig), and mouse anti-β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China) (dilution concentration 1:5000). HRP-conjugated goat anti-mouse (G1214) and goat anti-rabbit (G1213) were purchased from Servicebio (Wuhan City, China) (dilution concentration 2:5000). Alexa Fluor 568 goat anti-mouse IgG, IgM (H + L) (A-11004) and Alexa Fluor 647 goat anti-mouse-IgG (H + L) (A-21445) were purchased from Thermo Fisher (Waltham, MA, USA) (dilution concentration 1:500). Immunofluorescence antibodies were diluted in PBS. Immunoblot antibodies were diluted in TBST.

Frozen liver tissue sections and cultured cells were fixed in 4% paraformaldehyde for 15 min, rinsed in 0.1% Tween 20 in phosphate-buffered saline, and incubated in blocking buffer (DAKO, Tokyo, Japan). The primary and secondary antibodies were diluted in 1% bovine serum albumin/phosphate-buffered saline and incubated with the cells for 1 h at 37°C. The slides were then mounted using DAPI, and the cells were viewed using an image analysis system (BIOREVO BZ-9000; KEYENCE, Osaka, Japan). The following primary antibodies were used: rabbit anti-LC3B, rabbit anti-ATG16L1, rabbit anti-phospho STAT3 Tyr705 (Cell Signaling Technology, Inc., Danvers, MA), and mouse anti-LAMP2 (Abcam, Cambridge, MA). The slides were then incubated with Alexa Fluor 488 (goat anti-rabbit) and Alexa Fluor 594 (goat anti-mouse)-conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

MEF, organoids and IEC samples were lysed in E1A buffer supplemented with sodium orthovanadate, sodium fluoride and cOmplete^™, EDTA-free Protease Inhibitor Cocktail (Roche) for 5–10 minutes on ice, spun down cold at maximum speed and protein concentration was measured with Bradford. 20–35 µg of protein was loaded on the gels and separated by SDS-PAGE (PAGE), transferred to nitrocellulose or PVDF (Millipore) membranes, and analyzed by immunoblotting. Proteins were detected with following antibodies: mouse anti-A20 (Santa Cruz sc-166692, 1:1000), rabbit anti-Atg16l1 (Cell Signaling 8089, 1:1000); rabbit anti-LC3 (MBL PM036, 1:1000); rabbit anti-p62 (MBL PM045, 1:2000), goat anti-IkBa (Santa Cruz sc-371-G, 1:1000); mouse anti-P-IkBa (Cell Signaling 9246, 1:1000); mouse anti-actin (MP Biomedicals 8691002, 1:10,000); mouse anti-HA (BioLegend 901501, 1:1000); mouse anti-Flag (Sigma F1804, 1:1000); rabbit anti-GST (Cell Signaling 2622, 1:1000); mouse anti-Tubulin (Sigma T4026, 1:40,000); mouse anti-GAPDH (Abcam Ab8245, 1:10,000); mouse anti-Atg16l1 (MBL 150-3, 1:2000); mouse anti-LC3 (MBL M186-3, 1:2000); mouse anti-UB (FK2; Millipore 4-263, 1:1000). As secondary antibodies, anti–rabbit, anti-mouse (GE Healthcare) or anti-goat (Santa Cruz)-HRP conjugates were used, and the signal was detected with enhanced chemiluminescence substrate ECL (Perkin Elmer).