Anti phospho jnk1 2

Macrophages were infected with Mtb for the indicated times. Cells were lysed with lysis buffer consisting of 10 mM Tris–HCl, 1 mM EDTA, 140 mM NaCl, 0.1% DOC, 0.1% SDS, and Triton X-100 containing complete protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). Western blotting was performed as previously described (27 (link)). Anti-phospho-GSK3β, anti-IκBα, anti-cathepsin D, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PI3K, anti-PI3K, anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-phospho-MEK1/2, anti-phospho-JNK1/2, anti-phospho-IκBα, and anti-IRAK-M were purchased from Cell Signaling Technology.

The following commercial primary antibodies were used: anti-phospho-ERK1/2, anti ERK1/2, anti-JNK1/2, anti-phospho-JNK1/2 and anti-phospho-c-Jun (all from Cell Signaling Technology, Danvers, MA, USA); anti-phospho-EGFR (Thermo Fisher Scientific, Rockford, IL, USA); anti-EGFR, anti-paxillin and anti-c-Jun (Santa Cruz Biotechnology, Heidelberg, Germany); and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA). Secondary antibodies were anti-rabbit IgG Horseradish peroxidase-linked F(ab’)2 I fragment (from donkey) (GE Healthcare, GE, Little Chalfont, United Kingdom), anti-mouse IgG₁ (BD Pharmingen, Beckton Dickinson, Franklin Lakes, NJ, USA), and Alexa Fluor 488 conjugated anti-mouse (from donkey) (Thermo Fisher Scientific, Rockford, IL, USA).

Minimal essential medium-alpha (α-MEM), fetal bovine serum (FBS), and TRIzol were purchased from Invitrogen (Carlsbad, CA). Hybond C membrane and ECL Western blotting detection system were from Amersham Biosciences (Buckinghamshire, UK). Recombinant human TNF-α and the anti-TNFR1 neutralizing antibody were from R&D System (Minneapolis, MN). Luciferase assay kit was from Promega (Madison, WI). Metafectene transfection reagent was from Biontex Lab (GmbH, Planegg/Martinsried, Germany). SMARTpool RNA duplexes corresponding to Src, TRAF2, ERK2, p38 MAPK, JNK2, and scrambled #2 siRNA were from Dharmacon Research Inc (Lafayette, CO). Anti-phospho-IKKα/β, anti-phospho-NF-κB p65 (Ser⁵³⁶), anti-phospho-c-Src, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, and anti-phospho-IκB-α antibodies were from Cell Signaling (Danver, MA). anti-NF-κB (p65), anti-lamin A, anti-TRAF2, anti-TNFR1, anti-c-Src, anti-ERK2, anti-p38, anti-JNK2, anti-IκB-α, and anti-sICAM-1 antibodies were from Santa Cruz (Santa Cruz, CA). The anti-GAPDH antibody was from Biogenesis (Boumemouth, UK). Actinomycin D (Act.D), cycloheximide (CHI), PP1, U0126, SB202190, SP600125, GM6001, MMP2/9 inhibitor, and Bay11-7082 were from Biomol (Plymouth Meeting, PA). Enzymes and other chemicals were from Sigma (St. Louis, MO).

Cell lysates were suspended in RIPA lysis buffer (Beyotime, Nantong, China) including Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined by BCA assay. Equal amounts of proteins (100μg) were separated on 10% or 12% SDS-PAGE, then transferred to nitrocellulose membranes (Pall, Glen Cove, NY, USA). After being blocked with 5% BSA for 1 hour, the filters were incubated with the primary antibodies at 4°C overnight and incubation with the secondary antibody for 1.5 hours, and the bound was detected with the ChemiDoc^TM XRS system (Bio-Rad, Hercules, CA, USA). The antibodies used are as follows: anti-SIP1, anti-GADD45G, anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-JNK1/2 (Thr183/185), anti-JNK1/2, anti-phospho-c-Jun (Ser63), anti-c-Jun (Cell Signaling Technology; CST, Danvers, MA,USA); anti-phospho-HSP27 (Ser82) (Upstate, Lake Placid, NY, USA); anti-HSP27 (Epitomics, Burlingame, CA, USA).

Protein samples were extracted from kidney tissue or cultured PMECs. Equal amounts of protein were subjected to SDS-PAGE. Binding of the primary antibody was then detected using peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG-HRP, Bio-Rad Laboratories) and enhanced chemiluminescence (Amersham), and the band densities were quantified via densitometry. The antibody source and the dilution used were as follows: rabbit anti-calpain1 and 2, anti-phospho-p38, anti-phospho-JNK1/2, anti-phospho-ERK1/2, anti-total p38, anti-total JNK1/2, anti-total ERK1/2, anti-iNOS, and anti-eNOS antibodies (all at a 1:1000 dilution, Cell Signaling). Rabbit anti-GAPDH (1:1000 dilution, Santa Cruz) was used as the internal control.

The following commercial primary antibodies were used: anti-phospho-ERK1/2, anti ERK1/2, anti-JNK1/2, anti-phospho-JNK1/2 and anti-phospho-c-Jun (all from Cell Signaling Technology, Danvers, MA, USA); anti-phospho-EGFR (Thermo Fisher Scientific, Rockford, IL, USA); anti-EGFR, anti-paxillin and anti-c-Jun (Santa Cruz Biotechnology, Heidelberg, Germany); and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA). Secondary antibodies were anti-rabbit IgG Horseradish peroxidase-linked F(ab’)2 I fragment (from donkey) (GE Healthcare, GE, Little Chalfont, United Kingdom), anti-mouse IgG₁ (BD Pharmingen, Beckton Dickinson, Franklin Lakes, NJ, USA), and Alexa Fluor 488 conjugated anti-mouse (from donkey) (Thermo Fisher Scientific, Rockford, IL, USA).