Nf κb

PE-conjugated anti-chicken IgA (Cat No.8330–09, SouthernBiotech, Birmingham, AL, USA), AF647-conjugated anti-chicken IgM (Cat No.8310–31, SouthernBiotech), NF-κB (Cat No.bs-0465R, Bioss, Beijing, China), PE-conjugated anti-chicken MHC II (Cat No.8345–09, SouthernBiotech), β-actin (Cat No.AC026, Abclonal), P70S6K1 (Cat No.A2190, Abclonal), phospho-P70S6K1 T389 (Cat No.AP1059, Abclonal), Goat anti-rabbit IgG (H + L) Alexa Fluor 594 (Cat No.A11012, Invitrogen), GW9662 (Cat No.HY-16578, MCE, Shanghai, China), S3I-201 (Cat No.SD4794, Beyotime Biotechnology, Shanghai, China).

Cell lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% BSA for 1 h, then incubated with primary antibodies overnight at 4 °C. Antibodies were used as following: ERCC6L (Proteintech, 15,688–1-AP, 1:1000), PI3K (Bioss, bs-0128R, 1:1000), p-PI3K (Ser1070; Bioss, bs-6417R, 1:1000),AKT1 (Bioss, bs-0115R, 1:1000), p-AKT1 (Thr34; Bioss, bs-5194R, 1:1000), JAK2 (Bioss, bs-23003R, 1:1000), p-JAK2 (Tyr1007 + Tyr1008; Bioss, 2485R, 1:1000), NF-κB (Bioss, bs-0465R, 1:1000), p-NK-κB (Thr505; Bioss, bs-5663R, 1:1000), and β-actin (Bioss, bs-0061R, 1:5000). The next day membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. After washed for 3 times in TBST for 5 min, membranes were visualized by chemiluminescence kit and scanned with QuantityOne software (Bio-Rad, Hercules, CA, USA). The bands were analyzed with ImageJ (NIH, Bethesda, MA, USA).

Total protein was extracted by lysis buffer for Western blotting with 100 mM of phenylmethanesulfonylfluoride, and 25 μg of total protein sample was subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% methanol at 4 °C. The membranes were blocked with 5% skim milk for 2 h, following incubated overnight with diluted primary antibodies against Bcl-xl (1:5000, Abcam, Cambridge, UK), caspase 3 (1:2000, Cell signaling technology, Boston, MA, USA), Bip (1:2000, Cell signaling technology), PERK (1:5000, Abcam), p-PERK (1:1000, Bioss), ATF4 (1:2000, Proteintech, Chicago, IL, USA), ATF6 (1:2000, Proteintech), eIf2α (1:1000, Proteintech, Wuhan, China), p-IRE1(1:1000, Bioss), CHOP (1:1000, Bioss), NF-κB (1:1000, Bioss), IκB (1:2000, Cell signaling technology), ZO-1 (1:1000, Bioss, Beijing, China), Occludin (1:1000, Bioss), Claudin-1 (1:1000, Bioss), and GAPDH (1:1000, Proteintech, China) followed by goat anti-rabbit IgG (H+L; 1:1000, Proteintech, China). The gray values of protein bands were measured by ImageJ software version 6.1 (Bio-Rad Laboratories, Hercules, CA, USA). The cellular protein PERK was used as an internal control for p-PERK, and GAPDH was used as an internal control for other proteins.