Anti phospho perk

Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l-cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell lysates and brain homogenates were prepared in PBS supplemented by a cocktail of protease inhibitors. Protein concentration was determined with the Pierce BCA Protein Assay kit (Thermo Scientific). Proteins from cell lysates or brain homogenates were fractionated by electrophoresis using 4–12% SDS-polyacrylamide gels (SDS-PAGE), transferred into nitrocellulose membranes, and probed with the following antibodies: anti-prion 6D11 antibody (1:5000, Sigma), anti-calreticulin (1:1000, Cell Signaling), anti-GRP94 (1:1000, Cell Signaling), anti-protein disulphide isomerase (anti-PDI, 1:1000, Cell Signaling), anti-protein kinase RNA-like endoplasmic reticulum kinase (PERK) (1:1000, Cell Signaling), anti-phospho-PERK (1:1000, Cell Signaling), anti-inositol-requiring 1 protein (IRE1) (1:1000, Cell Signaling), anti-eIF2α (1:1000, Cell Signaling), anti-phospho-eIF2α (1:1000, Cell Signaling), anti-calnexin (1:1000, Cell Signaling), anti-CCAAT/-enhancer-binding protein homologous protein (CHOP) (1:1000, Cell Signaling), and anti-β-actin (1:1000, Cell Signaling). The immunoreactive bands were analyzed using the Quantify One (4.6.7) software (BioRad^®).

After treatment as indicated, the cells were washed with cold 1 × PBS, lysed with RIPA lysis buffer for 20 min on ice, and centrifuged at 12,000 r.p.m. for 10 min at 4 °C. Protein was denatured at 95 °C for 5 min. Samples (20 µg each) were analyzed by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 1% Tween-20 for 1 h and incubated with corresponding primary antibodies at 4 °C overnight. Then, the membranes were washed with Tris-buffered saline containing Tween-20 three times for 5 min each and incubated with corresponding secondary antibodies for 1 h at room temperature. Protein brands were detected by chemiluminescence (Millipore).
The primary antibodies anti-Bax (ab32503), anti-Bcl-2 (ab692), anti-ATF4 (ab184909), anti-CHOP (ab11419), anti-eIF2A (ab169528), anti-eIF2S (ab32157), and anti-PERK (ab79483) were purchased from Abcam (Cambridge, UK). Anti-phospho-PERK was purchased from Cell Signaling Technology (Beverly, MA, USA). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA): goat anti-rabbit IgG (H&L), goat anti-mouse IgG (H&L), and rabbit anti-goat IgG. LBP was purchased from Changan Hainan International Pharmaceutical Co., Ltd (Haikou, Hainan, China).

Cell lysates prepared from macrophages were subjected to western blotting. The membranes were probed with the following primary antibodies: anti-GRP78, anti-phospho-eIF2α, anti-CHOP, anti-caspase-3, anti-phospho-PERK, and anti-PERK (all from Cell Signaling Technologies, Danvers, MA, USA). Additional antibodies included anti-CRT, anti-IRE1α, anti-ATF-6α (Santa Cruz Biotechnology), anti-CXCR1 (Biorbyt, San Francisco, CA, USA), and anti-ERp57 (Abcam, Cambridge, MA, USA). The primary antibodies were used at a 1:1000 dilution. The secondary antibodies consisted of goat anti-rabbit IgG (Cell Signaling), goat anti-mouse IgG (Santa Cruz Biotechnology), and rabbit anti-goat IgG (Calbiochem, San Diego, CA, USA). β-actin was used as the loading control (Santa Cruz Biotechnology). Macrophages were pretreated with inhibitors or inducers for 1 h prior to Mtb H37Ra infection. Specific inhibitors of JNK (SP600125), ERK (PD098059), and p38 (SB203580) were purchased from Calbiochem and NAC, a specific inhibitor of ROS, from Sigma-Aldrich (MO, USA). GSK2606414 (Calbiochem), Irestatin (Axon Medchem, Groningen, Netherlands), and AEBSF (Sigma-Aldrich) were used as selective inhibitors of PERK, IRE1α, and ATF6, respectively.

The following antibodies were used: anti-GRP78, anti-GRP94, anti-ATF6, anti-PERK, anti-phospho-PERK, anti-eIF2α, and rabbit anti-phospho-eIF2α antibody (Cell Signal Technology, Inc., Danvers, MA, USA). The rabbit antibodies against LC3 and p62 and a mouse monoclonal antibody against HA and β-actin, the secondary antibodies tetramethyl rhodamine isothiocyanate TRITC-conjugated goat anti-mouse and horseradish peroxidase conjugated goat anti-mouse or anti-rabbit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Beclin1 rabbit antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-VP1 mAb was prepared in our laboratory. Tunicamycin (Tu) and thapsigargin (Tg) were purchased from Sigma-Aldrich. RIPA lysis buffer was obtained from Beyotime (Jiangsu, China). Lipofectamine LTX and PLUS and RNAiMAX reagent were purchased from Invitrogen (Auckland, NY, USA).

Following the treatments, the cell lysates were prepared with TEGN buffer (10 mm Tris-HCl pH 7.5, 1 mm EDTA, 400 mmNaCl, 0.5% NP-40 and 1 mm DTT) supplemented with protease inhibitor cocktail. Western blotting was carried out as described.^{41 (link)} The antibodies used—anti-phospho-JNK (#4668S), anti-phospho-AKT (#9271S), anti-phospho-PERK (#3191S), anti-eIF2α (#9722S), anti-phospho-eIF2α (#3597S), anti-Bcl2 (#2872S), cleaved-caspase-3 (#9661S) were purchased from Cell Signalling Technologies, Beverly, MA, USA; pro-caspase-3 (ab44976, Abcam, Cambridge, UK); anti-Par-4 (sc-1807), anti-p21 (sc-817), anti-AKT (sc-8312), anti-PERK (sc-13073), anti- FAS (sc-714), anti-CDK2 (sc-748), anti-XIAP, anti-PARP (sc-7150), anti-GRP78 (sc-13968), anti-JNK (sc-572), p53 (sc-6243), anti-GAAD (sc-575) antibody were from Santa Cruz Biotechnology, Inc. Anti-β-actin (A5316), anti-Rabbit (A6154) and anti-Mouse (A4416) monoclonal antibody were purchased from Sigma-Aldrich. The quantitative analysis of western blot results was based on ImageJ software version 1.44p (NIH, USA). For co-immunoprecipitation, lysates were precleared by adding 20 μl of normal IgG conjugated to 50 μl of protein G-Sepharose beads. Twenty-five microliters of antibody conjugated to 50 μl of protein G-Sepharose beads was used to immunoprecipitate the precleared lysates.