Phospho-Jak1 (Tyr1034/1035) Antibody #3331

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	-Rabbit (1:1000)

Protocol tips
-Rabbit (1:1000)

Publication protocol

STATs' phosphorylation was tested in complemented U4A cells, U4C-FRT cells or patient fibroblasts along with appropriate control cells upon stimulation with IFN-α (11101–2, PBL) or IFN-γ (IF002, Millipore), in full growth medium at 37 °C. Then, the cells were trypsinized, washed and lysed in cold radioimmunoprecipitation assay buffer supplemented with phosphatases (P5726, Sigma) and protease inhibitors (11836153001, Roche) for 30 min at 4 °C. The lyses were then centrifuged at 10,000g for 10 min at 4 °C, the supernatants were quantified and 10–50 μg of total protein was separated by 10% SDS-PAGE and analysed by western blot. Membranes were cut horizontally according to molecular size markers, and stripes were incubated with different Abs. Immunoblots were developed with the enhanced chemiluminescence western blotting Reagent (Amersham). The following Abs were used: anti-JAK1 (610231, BD Biosciences; 1/1,000 dilution), anti-pJAK1 Tyr1022/1023 (3331, Cell Signaling Technology; 1/1,000 dilution), anti-JAK2 (Clone D2E12, 3230, Cell Signaling Technology; 1/1,000), anti-pJAK2 Tyr1007/1008 (Clone C80C, 33776, Cell Signaling Technology; 1:1,000), anti-TYK2 (Clone D4I5T, 14193, Cell Signaling Technology; 1:1,000), anti-pTYK2 Tyr1054/1055 (Clone C80C, 9321, Cell Signaling Technology; 1:1,000), anti-STAT1 (9172, Cell Signaling Technology; 1:1,000), anti-pSTAT1 Tyr701 (Clone 58D6, 9167, Cell Signaling Technology; 1:1,000), anti-STAT2 (4594, Cell Signaling Technology; 1:1,000), anti-pSTAT2 Tyr689 (07–224, Millipore; 1:2,000), anti-β-Actin (Clone AC-15, A5451, Sigma-Aldrich; 1:10,000), anti-GFP (11814460001, Roche or ab290, AbCam; 1:2,000) and anti-Myc (clone 9B11, 2276, Cell Signaling Technology; 1:1,000). Band densitometry was determined by Image Studio Lite (Licor). Fold changes of phospho/non-phosphorylated proteins were calculated against the indicated internal reference sample. Similarly, JAK1 protein levels in primary fibroblasts were determined after normalisation with β-actin (loading control).

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