Rabbit anti β actin

The cell lysates were harvested with 2× Laemmli SDS-PAGE sample buffer (Bio-Rad No. 1610737) containing a final concentration of 5% β-mercaptoethanol (Bio-Rad No. 1610710). The cell lysates were then denatured at 95°C for 10 min. The lysates were then loaded onto a Mini-PROTEAN TGX gel (Bio-Rad No. 4561096) and electrophoresed, followed by transfer to a polyvinylidene difluoride membrane (Bio-Rad No. 1620177). The membrane was then blocked in 5% nonfat dry milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h, followed by a short TBS-T wash. Overnight incubation with primary antibody, either rabbit anti-hIFIT1 (Cell Signaling Technology No. 14769) or rabbit anti-β-actin (Cell Signaling Technology No. 4970) was then performed. Afterward, the membrane was washed three times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology No. 7074) for 1 h. Finally, the membrane was washed three times with TBS-T, incubated with Clarity Western ECL Substrate (Bio-Rad No. 1705060) and imaged with a Bio-Rad ChemiDoc Imaging System running Bio-Rad Image Lab Touch software (version 2.4.0.03).

The monoclonal mouse anti-NS5A 9E10 antibody was generously provided by Charles Rice (Rockefeller University, NY). The following commercial primary antibodies were used: rabbit anti-FLAG for flow cytometry (1:1500, #14793S, Cell Signaling Technology); rabbit anti-human SEC14L2 for western blot (1:1000, catalog #LS-B11733, LifeSpan BioSciences, Inc); mouse anti-human CypA for western blot (1 µg/ul, catalog #58144, AbCam); human CD81 conjugated to PE monoclonal for flow cytometry (1:200, catalog #BDB555676, BD Biosciences), rabbit anti-β actin for western blot (1:2000, catalog #4970S, Cell Signaling Technologies), mouse anti-β actin for western blot (1:1000, catalog #3700S) and rabbit anti-human/mouse/rat/monkey CypA (catalog #2175S, Cell Signaling Technologies). The following commercial secondary antibodies were used: goat anti-mouse Alexa 647 (1:250, catalog #A-21235, Invitrogen) for flow cytometry; goat anti-rabbit Alexa 700 for flow cytometry (1:250, catalog #A-21038, Invitrogen); goat anti-mouse Dylight 800 for western blot (1:10,000, catalog #SA535521, Thermo Fisher Scientific); and goat anti-rabbit Dylight 680 for western blot (1:10,000, catalog #35568, Thermo Fisher Scientific).

After indicated treatment, cells were harvested and washed twice with PBS. Then they were lysed in RIPA buffer (Solarbio, Beijing, China) containing complete protease and phosphatase inhibitor (Solarbio, Beijing, China). The level of protein concentrations in cells was measured with a bicinchoninic acid protein assay kit (Solarbio, Beijing, China). The proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes by a wet electrophoretic transfer method. The membrane was blocked with TBST containing 5% non-fat milk for 2 h at room temperature followed by incubation overnight at 4 °C with primary antibodies. After overnight incubation, the blots were washed three times with TBST and incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were scanned using a Bio-Rad Gel imaging system (Bio-Rad, Berkeley, CA, USA) after visualization treatment with the ECL reagent. The primary antibodies, rabbit anti-PBK, rabbit anti-KIAA0101, rabbit anti-TOP2A, rabbit anti-IL-1β, and rabbit anti-β-actin, were purchased from Cell Signaling Technology (Boston, MA, USA). The rabbit anti-ASPM and rabbit anti-MCM10 were obtained from Proteintech (Rosemont, Chicago, IL, USA). Quantitative assessment of the and intensity was performed using Image Lab statistical software.

Cells specimens were lysed using RIPA lysis buffer (Boster, Wuhan, China). The equal amounts of total proteins were separated by 10% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, MA, USA). The membranes were blocked with 5% skim milk powder at room temperature for 1 h and incubated with primary antibodies diluted in blocking buffer at 4°C overnight. Subsequently, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected by an ECL chemiluminescence kit (Millipore) according to the manufacturer's instructions. Protein levels were calculated relative to β-actin. Primary antibodies were used as follows: rabbit anti-NFAT2 (phospho S237) (Abcam, Cambridge, UK, ab183023), rabbit anti-p53 (#2527), rabbit anti-Bax (#5023), rabbit anti-Bcl-2 (#4223), rabbit anti-cleaved-caspase-3 (#9664), rabbit anti-NFAT2 (#8032), and rabbit anti-β-actin (#4970) were all from cell signaling (Danvers, MA, USA).

At the end of the incubation periods, islets and INS-1E cells were washed in ice-cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with Protease- and phosphatase inhibitors (Pierce, Rockford, IL, USA). Protein concentrations were determined with the BCA protein assay (Pierce). Equivalent amounts of protein from each treatment group were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and electrically transferred onto PVDF membranes. After blocking by 2.5% milk (Cell Signaling) and 2.5% BSA, membranes were incubated overnight at 4 °C with rabbit anti-cleaved caspase-3 (#9664), rabbit anti-PARP (#9532), rabbit anti-cleaved PARP (rat specific #9545), rabbit anti-phospho YAP(S127) (#4911), rabbit anti-LATS2 (#5888), rabbit anti-tubulin (#2146), rabbit anti-GAPDH (#2118), rabbit anti-β-actin (#4967) (all Cell Signaling Technology), and rabbit anti-PDX1 (#47267) and rabbit anti-p-MST1 (#79199) (both from Abcam) antibodies, all at a dilution of 1:1000, followed by horseradish-peroxidase-linked anti-rabbit IgG (Jackson). Membrane was developed by using a chemiluminescence assay system (Pierce) and analyzed using DocIT^®LS image acquisition 6.6a (UVP BioImaging Systems, Upland, CA, USA). Uncropped and unprocessed scans of all Western blots are available in the Source Data file.