Hsp90

Antibodies used were HA (SC-805, Santa Cruz, Dallas, TX), HA-7-HRP (H6533, Sigma-Aldrich), MEK1/2 (#9126, Cell Signaling, Danvers, MA), phospho-MEK1/2 (#2338, Cell Signaling), ERK1/2 (M5670, Sigma-Aldrich), phospho- ERK1/2 (#4370, Cell Signaling), STAT5 (610191, BD Biosciences, Franklin Lakes, NJ), phospho-STAT5A/B (05–886R, Merck Millipore), phospho-p70 S6 kinase (#9234, Cell Signaling), p70 S6 kinase (SC-230, Santa Cruz), RIPK3 (#12107, Cell Signaling), HSP90 (610418, BD Transduction Laboratories), actin (AAN01-A, Cytoskeleton, Denver, CO), and tubulin (ab7291, Abcam, Cambridge, UK). The secondary antibodies used were goat anti-mouse HRP (115–035-003, Jackson ImmunoResearch, West Grove, PA), goat anti-rabbit HRP (111–035-003, Jackson ImmunoResearch), and Alexa Fluor 680 goat anti-mouse (A-21057, Molecular probes, Grand Island, NY).

Protein lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies raised against human Annexin A4 (R&D Systems, Cat. No.: MAB4146. Dilution 1:500), Annexin A6 (Abcam, Cat. No.: ab31026. Dilution: 1:1000), heat shock cognate 70 kDa protein (Hsc70; N69. Dilution: 1:5000. Kindly provided by Boris Margulis, Russian Academy of Sciences, St Petersburg, Russia), tGFP/tRFP (OriGene, Cat. No.: TA150041/TA150061. Dilution 1:1000), heat shock protein 90 (Hsp90: BD Transduction Laboratories, Cat. No.: 610418. Dilution: 1:4000), and β-actin (Sigma-Aldrich, Cat. No.: A2228. Dilution 1:5000) were used followed by appropriate peroxidase-conjugated secondary antibodies (DAKO). Immunocytochemistry: Cells on coverslips were fixed in methanol and stained with indicated primary antibodies (1:100 dilution) including Annexin A4 (R&D Systems). Samples were incubated with the appropriate Alexa Fluor-594-coupled secondary antibodies (1:1000 dilution) (Molecular Probes) and images taken by a Zeiss confocal microscope.

Cultured SMCs were washed with ice cold PBS twice, and 80 μl Laemmli sample buffer (60 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS) was added. After lysis and protein determination using a detergent-compatible protein assay from Bio-Rad (500–0116), bromophenol blue (0.01%) and β-mercaptoethanol (5%) were added to the remainder of the lysates. ≈20 μg protein was loaded per lane on Bio-Rad TGX Criterion gels and proteins were separated using Bio-Rad Tris/Glycine/SDS electrophoresis buffer at 200 V. Following separation, proteins were transferred to nitrocellulose (0.2 μm) using the Trans-Blot Turbo Transfer System (Bio-Rad) and western blotting was done essentially as described^{57 (link)}. Membranes were cut horizontally to allow for detection of multiple targets as needed, and hence blots covering the entire range of molecular weights are not available throughout. Full blots for all display items are available in the Supplementary Information file. The following primary antibodies were used: NEXN (Abcam, ab213628), P-YAP (Cell Signaling Technology,#4911), T-YAP (Cell Signaling Technology, #4912), HSP90 (BD Transduction Laboratories, 610418), calponin/CNN1 (Abcam, ab46794), SM22/TAGLN (Abcam, ab14106). Secondary anti-mouse and anti-rabbit antibodies were from Cell Signaling Technology (#7076 S, #7074 S).

RNA was extracted from osteoclasts using peqGOLD TriFast (PeqLab, Erlangen, Germany) at the indicated time points, reverse transcribed into cDNA and analyzed with SYBR Green-based quantitative Real-Time PCR in triplicates. mRNA expression level was normalized to GAPDH, β-actin or RNA polymerase II Polr2a. GAPDH is commonly used as a reference gene in osteoclast studies and indeed was stable in our experiments. To confirm that it does not affect the results when analyzing genes involved in energy metabolism we also used the mentioned above genes.
Antibodies to the following proteins were used for western blotting: NFATc1 (sc-7294, Santa Cruz), DC-STAMP (MABF39, Millipore), Sirt1 (07–131, Millipore), Sirt3 (#5490, Cell signaling), acetylated Sod2 (acetyl-superoxide dismutase K68, ab137037, Abcam), Sod2 (ab16956, Abcam), phosphorylated AMPKα (AMP-activated protein kinase, #2535, Cell Signaling), AMPKα (#2793, Cell Signaling), phosphorylated ACC (Acetyl CoA Carboxylase, #3661, Cell Signaling), ACC (#3662, Cell Signaling), acetylated p65 (Acetyl-NF-κB p65(Lys310) #3045, Cell signaling), p65 (#3987, Cell signaling), IκBα (#4814, Cell signaling), HSP90 (610419, BD Transduction laboratories) and GAPDH (ab8245, Abcam, UK) were used as reference proteins.