Chicken anti mouse igg hrp

Whole-cell lysates were extracted with cell culture lysis buffer (Promega, Madison, WI, USA), and protein concentrations were quantified with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The transfer membrane was blocked and probed with the following antibodies prepared in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-STMN1 (1:1000, 13655S, Cell Signaling, Danvers, MA, USA), anti-FLAG-M2-HRP (1:1000, A8592, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich). The membrane was then probed at room temperature for 1 h with the corresponding secondary antibodies: bovine anti-rabbit IgG-HRP (1:3000, sc-2370, Santa Cruz Biotechnology, Dallas, TX, USA) and chicken anti-mouse IgG-HRP (1:5000, sc-2954, Santa Cruz Biotechnology). Immunoblots were visualized in an ImageQuant LAS 4000 chemiluminescence detection system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). All Uncropped blots can be seen in Figure S8.

Protein extracts were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The transferred membrane was blocked and hybridized with the following antibodies in 5% BSA (Sigma-Aldrich) overnight at 4 °C: anti-ALIX (1:1000, 2171S, Cell Signaling), anti-TSG101 (1:1000, EXOAB-TSG101-1, System Biosciences), anti-CD81 (1:1000, GTX101766, Genetex), anti-Numb (1:1000, ab14140, Abcam), anti-β-catenin (1:1000, 610154, Becton-Dickinson), anti-CXCL1 (1:1000, AF-453-NA, R&D Systems Inc.), anti-CXCL2 (1:1000, AF-452-NA, R&D System Inc.), anti-FLAG tag (1:1000, F1804, Sigma-Aldrich), and anti-β-actin (1:5000, A5441, Sigma-Aldrich). The corresponding secondary antibodies were used for hybridization at room temperature for 1 h: bovine anti-rabbit IgG-HRP (1:3000, sc-2370, Santa Cruz Biotechnology), chicken anti-goat IgG-HRP (Catalog sc-2953, Santa Cruz Biotechnology), and chicken anti-mouse IgG-HRP (1:5000, sc-2954, Santa Cruz Biotechnology). Immunoblots was visualized with a chemiluminescence detection system (ImageQuant LAS 4000, GE Healthcare Bio-Sciences).

Immunohistochemistry was used to assess in situ GRP78 expression in paraffin-embedded lung tissue sections [13] (link), [21] (link). In brief, immunostaining was performed with a rabbit monoclonal antibody directed against Grp78 (Cell Signaling Technology, Danvers, MA) employing citrate antigen retrieval, as per the manufacturer's recommendation, biotinylated goat anti-rabbit IgG Jackson Immunoresearch West Grove, PA), and AB Complex HRP (Vector Laboratories, Burlingame, CA). Imaging methods have been previously detailed [13] (link), [21] (link).
GRP78 in BALF was detected by immunoblotting. Concentrated BALF (12 mcg protein) specimens were electrophoresed and processed as described above. Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA). This immunoblotting method was validated using both commercial rGRP78 and lysates of normal human CD14⁺-derived macrophages (Figure S2 in File S1).