Rabbit anti ampk

The cells were washed twice with ice-cold PBS and lysed in Cell Lysis Buffer (1x). Protein levels were determined using the Bradford method, and then the samples were mixed with Laemmli buffer and denatured at 95°C for 5 min. Equal amounts of proteins were separated on SDS/PAGE gels. All proteins were transferred to nitrocellulose membranes at 100 V. Membranes were washed for 5 min in TBS-Tween buffer (0.1% TBST) (100 mM Tris-buffered saline, 140 mM NaCl, and 0.1% Tween 20; pH 7.6) and the nonspecific bindings were blocked for 1 h at RT with 5% BSA in 0.1% TBST or with 5% nonfat milk solution in 0.1% TBST. Immunodetection was performed overnight at 4 °C using rabbit antiparkin (1:500; Cell Signaling), rabbit anti-AMPK (1:1000, Cell Signalling), rabbit anti-p-AMPK (1:1000, Cell Signalling), rabbit anti-Ulk-1 (1:200, Cell Signalling), rabbit anti-p-Ulk-1 (1:200, Cell Signalling), and antimouse total OXPHOS (1:500, Abcam) antibodies. Then, the membranes were washed three times (5 min) in TBST and incubated for 60 min at RT with antirabbit or antimouse secondary antibody (1∶4000) in a 5% nonfat milk/TBST. Antibodies were detected using chemiluminescent Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) under standard conditions. Immunolabeling of GAPDH (rabbit anti-GAPDH; 1:40,000; Sigma-Aldrich) for cell lysates was performed as a loading control.

Western blot analysis was performed as previously described [22 (link)]. Whole animal protein extract prepared from 300 gravid adult hermaphrodites from each treatment was used per gel well. Antibodies bound to a nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA) were visualized using an ECL Western blotting detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), and the respective band intensities were measured using a LAS-3000 image analyzer with Multi Gauge (v.3.0) (Fuji Film, Tokyo, Japan). The following primary and secondary antibodies were used: rabbit anti-AMPK (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pAMPK (1:1000, Cell Signaling Technology, Danvers, MA, USA), mouse anti-α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO, USA), HRP-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA).

Primary antibodies were mouse anti-PRRSV N protein (produced in our laboratory), mouse anti-β-actin (Proteintech, #A5316), mouse anti-Flag (Proteintech, #80010-1-RR), rabbit anti-LC3 (Proteintech, #14600-1-AP), rabbit anti-ATG7 (Cell Signaling Technology, #2631), rabbit anti-CaMKII (Abcam, #ab168818), rabbit anti-p-AMPK (Thr172) (Cell Signaling Technology, #2535), rabbit anti-AMPK (Cell Signaling Technology, #2532), rabbit anti-p-mTOR(Ser2448) (Cell Signaling Technology, #5536), rabbit anti-mTOR (Cell Signaling Technology, #2983), rabbit anti-STIM1 (Cell Signaling Technology, #4916), mouse anti-Orai1 (Proteintech, #66223–1), rabbit anti-GRP78 (Proteintech, #11587-1-AP) and rabbit anti-Calnexin (Abcam, #ab92573). HRP-labeled rabbit or mouse secondary antibodies were purchased from Beyotime (China).
Chemicals used included 2-Aminoethyl diphenylborinate (2-APB) (Selleck, #S6657), 3-MA (Selleck, #S2767), 4-Phenylbutyric acid (4-PBA) (Selleck, #S3592), BAPTA-AM (Selleck, #S7534), Dantrolene sodium (Selleck, #S5478), Dorsomorphin (Compound C) 2HCL (Selleck, #S7306), KN-93 (Selleck, #S7423), ML-9 HCL (Selleck, #S6847), Procaine (Selleck, #S4668), Rapamycin (MedChemExpress, #HY-10219), Tetracaine-HCL (Selleck, #S2573), Thapsigargin (Selleck, #S7895), Tunicamycin (Beyotime, #SC0393).