Mouse anti tubulin t9026

Cells were washed with ice-cold PBS, then lysed with 4xLSB-DTT. Lysates were briefly sonicated prior to boiling and loaded on SDS-PAGE gels. Trans-Blot turbo system (BioRad) was used for transfer. After 1 hour blocking (5% BSA), following antibodies were used for detection of proteins: 1:1000 mouse anti-Cnn3 (A-2, SCBT), 1:5000 mouse anti-tubulin (T9026, Sigma-Aldrich). Anti-mouse HRP secondary antibody (Invitrogen) and Western Bright ECL-spray (Advansta) were used for chemiluminescence detection of the protein bands.

H295R-S2 cells were lysed using RIPA buffer (Bio Basic Canada Inc.) with protease and phosphatase inhibitors mini tablets, EDTA free (Thermo Scientific). Proteins were solubilized for 30 min at 4 °C, under end-over-end rotation, and then centrifuged at 13,000 rpm for 15 min at 4 °C. Protein concentration was determined using Bradford protein assay (Biorad). 15 µg of proteins were submitted to 10% SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blotted with the following antibodies: mouse anti-aldosterone synthase antibody (1:500, clone CYP11B2-41-13, kindly provided by Dr C Gomez Sanchez^{92 (link)} and mouse anti-tubulin (T9026, 1:2000, Sigma Aldrich). Signals were developed by Clarity Max™ Western ECL substrate (Biorad, Hercules, CA) and detected by Fujifilm Las-4000 mini Luminescent image analyzer (Fujifilm, Tokyo-Japan) and quantified by Multi gauge software (Fujifilm, Tokyo-Japan). Expression of total proteins was normalized to the expression of the housekeeping protein tubulin. Uncropped and unprocessed scans of blots are presented in the Source Data file.

Rabbit polyclonal antibodies, ab28284 (against N-terminal end of ADAMTS-1), ab84792 (against C-terminal end of ADAMTS-4), ab41037 (against 600–700 residues of ADAMTS-5) and ab60148 (against catalytic domain of ADAMTS-20) were purchased from Abcam (Cambridge, UK) and Goat anti-rabbit IgG conjucated with peroxidase was from EMD Millipore Corporation (Billerica, Massachusetts, USA). Mouse anti-tubulin (T9026) and peroxidase conjugated anti-mouse IgG (A4416) were obtained from Sigma-Aldrich Inc (St Luis, USA). Expose Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (ab94710) was from Abcam. Total RNA was extracted from frozen tissue and cultured cells using the NucleoSpin Macherey-Nagel (Düren, Germany) extraction kit, following the manufacturer’s protocol. RT-PCR was obtained from Takara (Otsu, Shiga, Japan) One Step RT-PCR kit, used for fresh colon tissue and from Takara PrimeScript 1^st strand cDNA Synthesis kit and Finnzymes (Espoo, Finland) DyNAzyme II DNA Polymerase kit, used for cultured cells. Human Primers for all molecules were designed in the lab, using the PerlPrimer program. All other chemicals used throughout the study were of the best available analytical grade.