Rabbit anti pstat6

Cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM Tris pH8) with protease and phosphatase inhibitors. Protein amounts were estimated by BCA method (Thermo Scientific # 23227). Equal amounts of protein were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane. Western blot analysis was performed using the following primary antibodies: rabbit anti-pSTAT6 (#9361, Cell Signaling Technology), rabbit total anti-STAT6 (#9362, Cell Signaling Technology), rabbit anti-SATB1 (#3650, Cell Signaling Technology), anti-GATA-3 (ABCAm, ab106625), mouse anti-β-Actin (ac004, Abclonal), mouse anti-γ-Tubulin (#T6557, Sigma), and appropriate anti-mouse and anti-rabbit secondary antibodies. Densitometry analysis was performed using ImageJ (v 1.5.1) (32 (link)).

Proteins from BMDMs and ipsilateral cortical tissue were extracted using RIPA buffer, equalized, and loaded onto 5–20% gradient gels for SDS PAGE (Bio-Rad; Hercules, CA). Proteins were transferred onto nitrocellulose membranes and then blocked for 1 h in 5% milk in 1 × TBS containing 0.05% Tween-20 (TBS-T) at room temperature. The membrane was incubated in mouse anti-arginase 1 (N-20) (1:1000; BD Transduction Laboratories, San Jose, CA), rabbit anti-STAT6 (1:1000; Cell signaling, Danvers, MA), rabbit anti-pSTAT6 (1:1000; Cell signaling), mouse anti-STAT3 (1:1000; Cell signaling), rabbit anti-pSTAT3 (1:1000; Cell signaling), or mouse anti-β-Actin (1:5000; Sigma-Aldrich) overnight at 4 °C, then washed three times in TBS-T, and incubated in appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. Membranes were washed three times in TBS-T, and proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford, IL). Chemiluminescence was captured ChemiDoc™ XRS+ System (Bio-Rad), and protein bands were quantified by densitometric analysis using BioRad Molecular Imaging Software. The data presented reflects the intensity of target protein band normalized based on the intensity of the endogenous control for each sample (expressed in arbitrary units).

Cells or lung tissue samples were lysed with RIPA buffer containing protease inhibitors and protein concentrations determined by BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). Denatured proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Merck Millipore, IPVH00010). Blottings were incubated with primary Abs overnight at 4 °C and then with secondary Abs for 1 h at room temperature. The following primary antibodies were used: Mouse monoclonal anti-β-actin antibody (Sigma, A5441) (1 : 1000), Rabbit anti-C3 (Abcam ab200999) (1 : 500), Rabbit polyclonal to Histone H3 antibody (citrulline R2 + R8 + R17)-ChIP Grade (Abcam, ab5103) (1 : 500), and Rabbit anti-pSTAT6 (Cell Signaling Technology 56554) (1 : 500).