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Western blotting and phosphorylation assay

MW Mengrui Wu
WC Wei Chen
YL Yun Lu
GZ Guochun Zhu
LH Liang Hao
YL Yi-Ping Li
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Cell lysates preparation and SDS–polyacrylamide gel electrophoresis /western blotting analysis were carried out according to a standard protocol10,45. For the phosphorylation assay, BMMs and pre-osteoclasts (derived from BMMs with 2-day treatment of RANKL and M-CSF) were starved for 5 h and stimulated with RANKL or M-CSF for different times. Proteins were harvested in cell lysis buffer supplemented with proteinase inhibitor cocktail (Sigma-Aldrich Co. LLC, 1:100) and phosphatase inhibitor cocktail 2 (P-5726, Sigma-Aldrich Co. LLC, 1:100). To extract nuclear and cytoplasmic proteins, NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific Inc) was used. Western blot was performed using following antibodies:, anti-p84 mouse IgG (GeneTex GTX70220, 1:1,000), anti-IκBα rabbit IgG (Cell Signaling Technology 4812, 1:1,000), anti-phospho-IκBα (Ser32) rabbit IgG (Cell Signaling Technology 2859, 1:1,000), anti-p38 rabbit IgG (Cell Signaling Technology 9212, 1:1,000), anti-phospho-p38 (Thr180/Tyr182) rabbit IgG (Cell Signaling Technology 4631, 1:1,000), anti-Erk rabbit IgG (Cell Signaling Technology 4370, 1:1,000), anti-phospho-Erk (Thr202/Tyr204) rabbit IgG (Cell Signaling Technology 4695, 1:1,000), anti-Akt rabbit IgG (Cell Signaling Technology 4685, 1:1,000), anti-phospho-Akt (Thr308) rabbit IgG (Cell Signaling Technology 2965, 1:1,000), anti-JNK rabbit IgG (Cell Signaling Technology 9251, 1:1,000), anti-phospho-JNK (Thr183/Tyr185) rabbit IgG (Cell Signaling Technology 9258, 1:1,000), anti-c-Jun rabbit IgG (Cell Signaling Technology 9165), anti-phospho-c-Jun (Ser73) rabbit IgG (Cell Signaling Technology 9164, 1:1,000), anti- GSK3β rabbit IgG (Cell Signaling Technology 9315, 1:1,000), anti-phospho-GSK3α/β (Ser21/9) rabbit IgG (Cell Signaling Technology 9331, 1:1,000), anti-Gα13 rabbit IgG(Santa Cruz sc-410, 1:500), anti-Ctsk mouse IgG (Santa Cruz sc-48353, 1:500), anti-NFATc1 mouse IgG (Santa Cruz sc-7294, 1:500), anti-GAPDH mouse IgG (Santa Cruz sc-166574, 1:500), anti-β-Actin mouse monoclonal IgG (Santa Cruz sc-81178, 1:500), Goat anti-rabbit IgG-HRP (Santa Cruz sc-200416, 1:2,500), Rabbit anti-mouse IgG-HRP (Santa Cruz sc358917, 1:2,500). Full images of western blots are presented in Supplementary Fig. 10.

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