Gradient gel

HeLa cells with endogenous FLAG-tagged KCTD7 were harvested and lysed in lysis buffer [20 mM tris-Cl (pH 7.4), 100 mM NaCl, 0.2 mM EDTA, 0.5% NP-40, and 1× protease inhibitor cocktail]. After centrifugation, the WCLs were incubated with anti-FLAG antibody-conjugated M2 agarose beads (Sigma-Aldrich) at 4°C overnight to capture FLAG-tagged KCTD7. The beads were washed five times with lysis buffer and eluted with FLAG peptide. Then, the eluted proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) on a gradient gel (Bio-Rad) for Coomassie blue staining. Protein bands were excised from the gel and subjected to sequencing by MS.

Bacteria from plates were resuspended to an OD of 0.05 and incubated for 3 h at 37°C and 5% CO₂ under shaking conditions. Samples were taken, centrifuged and resuspended in 1 × sample buffer containing β-mercaptoethanol. The samples were heated at 95°C for 5 min. Proteins were separated on a gradient gel (4–15%, Bio-Rad) and transferred to an Immobilon-P membrane. For detection of PilE, a rabbit polyclonal antibody (1:5000) (Sjolinder and Jonsson, 2007 (link)) was used as a primary antibody, and IR-reactive dye conjugated rabbit antibody was used as a secondary antibody. The membrane was stripped to remove antibodies, and EF-Tu monoclonal mouse antibody (Hycult Biotech) was used together with IR-reactive dye conjugated anti-mouse antibody as a loading control. Blots were imaged with Odyssey IR scanner at 700 and 800 nm. For quantification, Image J (version 1.48) was used.

Cell lysates were prepared using RIPA lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were quantified by the BCA protein assay (Thermo Scientific, Rockford, IL, USA). Fifty micrograms of lysate was loaded onto a gradient gel (BioRad, Hercules, CA, USA) and subjected to gel electrophoresis. Semi-dry protein transfer to a PVDF nitrocellulose membrane (BioRad) was performed using the Transblot Turbo transfer system (BioRad). The membrane was then blocked in Odyssey blocking buffer (LiCOR, Lincoln, NE, USA) for 30 min and incubated with the appropriate antibody overnight at 4°C. Antibodies included E-cadherin (BD Biosciences, San Jose, CA, USA), β-actin (Abcam, Cambridge, MA, USA), Complex II Western Blot Antibody Cocktail that detects SDHB (30 kDa) and Complex Va (60 kDa) (Abcam, Burlungame, CA, USA), Methyl-Histone H3 Antibody Sampler Kit (Cell Signaling, Danvers, MA, USA), and SDHB (Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were then washed with TBS-T, incubated with the appropriate secondary antibody for 1 h at room temperature and then washed with TBS-T. The membrane signal was subsequently analyzed by the LiCOR Odyssey system (LiCOR Biosciences, Frankfurt, Germany).

After incubation with or without strontium at 12.5 μM for 7 days, 40 μg of total protein was separated by SDS-PAGE, using a gradient gel ((10–12%), Bio-Rad Laboratories), transferred to nitrocellulose membrane, and analyzed by immunoblotting using the chemiluminescence (Santa Cruz, CA, USA). The primary antibodies used were sclerostin (Abcam, MA, USA, 1 : 500) or GAPDH (Santa Cruz, CA, USA, 1 : 1000), peroxidase-conjugated anti-mouse IgG (Santa Cruz, CA, USA, 1 : 1000). ImageJ software was applied to compare the intensity of protein bands to control through quantifying.

Cells in culture were collected via trypsinization and pelleted via centrifugation. Cell pellets were lysed in AZ lysis buffer (50mM Tris pH 8, 250mM NaCl, 1% NP-40, 0.1% SDS, 5mM EDTA, 10mM Na4P2O7, 10mM NaF, 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche), 1x PhosSTOP (Roche)). The protein concentration of each sample was determined using the DC™ (detergent compatible) protein assay (Bio-Rad Laboratories, Inc.). Protein concentrations were normalized and samples were prepared with 5x Laemmli sample buffer. Samples were run on a gradient gel (Bio-Rad) and transferred for Western blot on 0.45um Nitrocellulose membrane (Bio-Rad).
The primary antibodies used were mouse anti-PTEN (sc7974, Santa Cruz Biotechnology), and mouse anti-Beta Actin (Cell Signaling Technology #4970). Primary antibodies were used at 1:1000 dilutions and were incubated for 1 to 2 hours at room temperature or overnight at 4°C. Secondary goat anti-mouse antibody (Thermo Fisher Scientific/Pierce) or were used at a 1:10,000 dilution for 1 hour at room temperature. Primary and secondary antibodies were prepared in 5% milk. Three washes with Tris Buffered Saline with Tween 20 were each performed after primary incubation and after secondary incubation. Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).