Anti nrp1

Cells (3 × 10⁵) were plated into six-well culture plates to achieve 70%–80% confluency, and were washed in PBS and suspended in 100 μL of RIPA buffer (Pierce, Dallas, TX, USA). Supernatant protein concentrations were determined using the BCA protein assay kit (Pierce). Supernatant samples containing 30 μg total protein were resolved by 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) depending on the molecular weights of the target proteins, and were transferred to immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by electroblotting, and then probed with anti-E-cadherin (Cat# sc-21791, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-N-cadherin (Cat# sc-53488, Santa Cruz Biotechnology), anti-Vimentin (Cat# gtx100619, GeneTex, Irvine, CA, USA), anti-SNAIL1 (Cat# gtx125918, GeneTex), anti-Twist (Cat# gtx127310, GeneTex), anti-ZEB1 (Cat# gtx105278, GeneTex), anti-STC2 (Cat# sc-14352, Santa Cruz Biotechnology), anti-ANXA2 (Cat# sc-9061, Santa Cruz Biotechnology), anti-NRP1 (Cat# 3725, Cell Signaling Technology, Danvers, MA, USA), anti-SMAD2 (Cat#: 3103, Cell Signaling Technology), or anti-GAPDH (Cell Signaling Technology) antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Merck Millipore, Billerica, MA, USA).

Total protein extracts of the medulloblastoma cell lines before and after expression of miR-148a, were separated by SDS-PAGE electrophoresis and blotted onto a PVDF membrane (Amersham-GE Healthcare Life Sciences, Mumbai, India). The membrane was then incubated with anti-NRP1 (#3725), anti-DNMT1 (#5119), anti-ROCK1 (#4035) antibody (Cell signaling technologies, Boston, MA, USA) or anti-γ-tubulin (T3559) antibody (Sigma-Aldrich, MO, USA) as per the manufacturer's instructions, followed by incubation with anti-rabbit IgG conjugated with Horse Radish Peroxidase and developed using SuperSignal West Pico chemiluminescent substrate (Pierce, Thermo Fischer scientific, Waltham, MA, USA). Protein expression levels were determined by intensity measurements of the bands corresponding to the proteins of interest using ImageJ software (http://imajeJ.nih.gov.in).

We performed western blotting analysis to validate the cross reactivity of the primary antibodies, using lysates from the MCT‐1 cell line developed in our laboratory from a grade III MCT derived from a 7‐year‐old male castrated Shar‐pei dog. The blots were blocked with 3% bovine serum albumin in TBS‐T (10 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 0.1% Tween‐20) for 2 h and incubated overnight with the following primary antibodies: mouse monoclonal anti‐α‐tubulin (diluted 1:400 000; Sigma‐Aldrich, Oakville, ON, Canada), rabbit monoclonal anti‐VEGFR2 (1:2000), anti‐phospho‐VEGFR2 (1:2000) or anti‐NRP‐1 (1:1000), or rabbit polyclonal anti‐c‐CBL (1:1000); all from Cell Signaling Technology, Danvers, MA, USA. Membranes were washed three times for 5–10 min in TBS‐T and incubated in HRP‐labelled goat polyclonal anti‐mouse or anti‐rabbit secondary antibodies (both at 1:20 000; Sigma‐Aldrich) for 1 h. Membranes were incubated with chemiluminescent HRP substrate Luminata™ Forte (EMD Millipore, Darmstadt, Germany) and bands were visualized in a Bio‐Rad ChemiDocTM XRS+ system (Bio‐Rad Laboratories Canada Inc., Mississauga, ON, Canada).