Fig. 5. SR3677 activates HK2 and increases its abundance at the mitochondria.

a HeLa cells were pre-treated with either DMSO or 0.5 µM SR3677 for 2 h prior to addition of 10 µM CCCP for 1 h. Immunostaining was performed against HK2. Scale bars, 10 µm. b The proportion of cells with HK2 localized to mitochondria was quantified (n = 3 independent experiments). Data are expressed as mean ± s.e.m. c SH-SY5Y cells treated with DMSO or 0.5 µM SR3677 were separated into total cell, cytosolic and mitochondrial fractions. Fractions were separated by SDS-PAGE and immunoblotting was performed with anti-HK2 and anti-CValpha antibodies. d Densitometry analysis was performed to quantify HK2 levels in each sample, followed by normalization to mitochondrial marker, CValpha. Average HK2/CValpha ratios are plotted (n = 2 independent experiments). e Serum-starved cells were treated with either DMSO or SR3677 for the indicated time (minutes) prior to harvesting. Lysates were separated by SDS-PAGE and immunoblotting was performed with anti-HK2 and anti-actin antibodies. Samples were also run on Phos-tag gels in parallel and immunoblotting was performed with anti-HK2 antibody. f Densitometry analysis was performed on the band indicated by the arrow in e. The average intensity of the indicated band normalized to actin loading control is expressed ± s.e.m. (n = 3 independent experiments). P-values were determined by paired Student’s t-test for b, f, *P < 0.05. All error bars represent s.e.m.