Anti pcna

The proteins from cell lysates (30 μg) extracts were denatured by heating. Then, they were resolved by 10% SDS-PAGE, and blotted onto a nitrocellulose membrane (BioTrace/NT) overnight in 50 mM Tris–HCl, 380 mM glycine, 0.1% SDS, and 20% methanol. Rabbit anti-PCNA (Life Technologies) (1:20,000), anti-p53 (Sigma-Aldrich) (1:2,000), anti-Bax or anti-Bcl-2 (Santa Cruz Technologies) (1:1,000) antibodies were then added and incubated for 1 h at room temperature. After treatment for 1 h at room temperature with the corresponding secondary antiserum (1:4,000 for anti-rabbit or anti-mouse sera), the signals were detected with enhanced chemiluminescence reagent (Pierce) using β-actin antibody as loading control.

Antibodies used were anti-Plk1 (EMD Millipore, #05-844), anti-GAPDH (Ambion, #AM4300), anti-p-paxillin (S272) (Sigma, #SAB4301321), anti-paxillin (BD Biosciences, #610052), anti-α-tubulin (Santa Cruz Biotech., SC-32293), anti-pericentrin (EMD Millipore, #ABT59), anti-γ-tubulin (Santa Cruz Biotehc., SC-17787), anti-CEP192 (Proteintech, #18832-1-AP), anti-p-paxillin (Y31) (ThermoFisher, #44-720G), anti-paxillin (Y118) (ThermoFisher, #44-722G) and anti-PCNA (ThermoFisher, #RB-9055-P). Paxillin siRNA (SC-37007) and control siRNA (SC-29439) were purchased from Santa Cruz Biotechnology.

The renal slides (4 μm thickness) were deparaffinized to distilled water and incubated with citrate buffer for antigen microwave retrieval. The slides were then washed with phosphate-buffered saline (PBS) and blocked using PBS containing 5% bovine serum albumin (BSA). The slides were incubated with primary antibodies including anti-DEC2 (1:200) and anti-PCNA (1:200, both from ThermoFisher Scientific, Waltham, MA, USA) overnight in a wet box at 4 °C. The slides were then incubated with corresponding HRP-conjugated secondary antibodies (ThermoFisher Scientific) at room temperature for 1 h and followed by diaminobenzidine and hematoxylin staining.

Salivary gland tissue was embedded in paraffin as previously described (May et al., 2015). Immunofluorescence and in situ hybridisation was performed as previously described (Gaete et al., 2015). Primary antibodies and dilutions were used as follows: anti‐Fgf10 (Rabbit) 1:500 (ABN44, Millipore); anti‐Sox9 (Rabbit) 1:300 (AB5535, Millipore) and anti‐Mist1 (mouse) 1:100 (sc‐98771, Santa Cruz Biotechnology) which was used with the TSA kit for signal amplification (PerkinElmer), anti‐beta‐Galactosidase (chicken) 1:500 (Ab9361 Abcam), anti‐Ecadherin (mouse) 1:100 (ab76055 Abcam), anti‐RFP (rat) 1:200 (5F8 Chromotek) and anti‐PCNA (mouse) 1:100 (Thermofisher Scientific). All immunoresults were analysed on a confocal laser‐scanning microscope (TCS SP5, Leica), including negative and positive controls. Z stacks were created to confirm expression levels of Sox9 within the nucleus. Confocal images were processed using Image J and Photoshop software.