3.0 × 105 HEK293 cells were seeded in each well of a six-well
plate (60–70% confluence) 24 h prior to transfection of the
microRNA (miR-215) expression vector (4 μg). Forty-eight hours
post-transfection, cells were lysed in lysis buffer (0.1m Tris,
150 mm NaCl, 10 mm EDTA, 0.2% Triton
X-100, 1% PMSF, protease inhibitor cocktail) at 4 °C for
10 min and centrifuged at 10 000 g for 30 min
at 4 °C, allowing cell debris to be pelleted and discarded. Western
blotting was performed as described previously.41 (link) Proteins were detected using primary antibodies for
60 min at room temperature or at 4 °C overnight. Secondary
antibodies were applied for 30–60 min at room temperature. Antibodies
were used as follows: ARHGEF39 (rabbit polyclonal, catalogue #131551,
manufacturer NovoPro Bioscience, Shanghai, China) at 1/2000 concentration,
B-actin (mouse monoclonal, catalogue #A5441, manufacturer Sigma-Aldrich) at
1/2000 concentration, secondary anti-mouse HRP-conjugated antibody (goat,
catalogue #1706516, Bio-Rad Laboratories, Hercules, CA, USA) at 1/5000
concentration and secondary anti-rabbit HRP-conjugated antibody (donkey
polyclonal, catalogue #16284, Abcam, Cambridge, UK) at 1/5000
concentration. Densitometry was performed using the Imagelab 5.2.1 (Bio-Rad)
program to calculate arbitrary units reflecting relative protein expression levels
for ARHGEF39 and β-actin.
plate (60–70% confluence) 24 h prior to transfection of the
microRNA (miR-215) expression vector (4 μg). Forty-eight hours
post-transfection, cells were lysed in lysis buffer (0.1
150 m
X-100, 1% PMSF, protease inhibitor cocktail) at 4 °C for
10 min and centrifuged at 10 000 g for 30 min
at 4 °C, allowing cell debris to be pelleted and discarded. Western
blotting was performed as described previously.41 (link) Proteins were detected using primary antibodies for
60 min at room temperature or at 4 °C overnight. Secondary
antibodies were applied for 30–60 min at room temperature. Antibodies
were used as follows: ARHGEF39 (rabbit polyclonal, catalogue #131551,
manufacturer NovoPro Bioscience, Shanghai, China) at 1/2000 concentration,
B-actin (mouse monoclonal, catalogue #A5441, manufacturer Sigma-Aldrich) at
1/2000 concentration, secondary anti-mouse HRP-conjugated antibody (goat,
catalogue #1706516, Bio-Rad Laboratories, Hercules, CA, USA) at 1/5000
concentration and secondary anti-rabbit HRP-conjugated antibody (donkey
polyclonal, catalogue #16284, Abcam, Cambridge, UK) at 1/5000
concentration. Densitometry was performed using the Imagelab 5.2.1 (Bio-Rad)
program to calculate arbitrary units reflecting relative protein expression levels
for ARHGEF39 and β-actin.