Anti jak1 rabbit monoclonal antibody

Archived BM paraffin blocks were cut into 5µM sections and fixed on slides. Microwave antigen retrieval was done in the presence of 1 mmol/L EDTA (pH 8.0) buffer. Slides were then incubated with anti‐CD138 (Cat# ms‐1793‐3, ThermoFisher, 1:20 dilution), anti‐JAK1 rabbit monoclonal antibody (#3344, Cell Signaling Technology, 1:100), or anti‐JAK2 antibody (#3230, Cell Signaling Technology, 1:50) for 30 minutes at room temperature, followed by Horseradish Peroxidase (HRP) labelled polymer antibody (DAKO, cat# K4001). Breast cancer tissue was used as the positive control for JAK1 and JAK2 staining and for optimization of antibody dilution and staining conditions (Figure S2).

Zirconia balls were added to the proteins extracted from the liver of P0 newborns. Then, the proteins were dissolved in 1 ml sample buffer (NuPAGE LDS sample buffer 250μL, sample reducing agent 100μL, 25×protease inhibiter 40μL, and water 610μL) and crushed. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant of each sample was subjected to SDS-PAGE. Strips of membrane were incubated with anti-p-JAK1 (Tyr1021), anti-JAK1 (ab125051; Abcam), anti-p-STAT1 (Tyr701), anti-STAT1 (ab99415; Abcam), anti-p-STAT3 (Tyr705), anti-STAT3 (SAB4300708, Sigma Aldrich), anti-p-STAT5 (Tyr694), anti-STAT5 (ab16276; Abcam), anti-p-STAT6 (Tyr641), anti-STAT6 (ab32520; Abcam) or anti-GAPDH (ab9485; Abcam) antibodies. The antibody–antigen complexes were detected with horseradish peroxidase–conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) at a dilution of 1:1,000, followed by detection with enhanced chemiluminescence Western blotting substrate (GE Healthcare BioSciences, Little Chalfont, UK), as described by the manufacturer. n=3. Each experiment was performed three times. For HEK293 cell lysates, additional anti-HaloTag monoclonal antibody (G9211; Promega Corporation, WI) and anti-JAK1 rabbit monoclonal antibody (#3344; Cell Signaling Technology, MA) were used as the primary antibodies.