HPS6 knockdown in HUVECs treated with HPS6 siRNA. HPS6 in HUVECs 48 and 72 hours posttransfection with HPS6 or control siRNA were detected by SDS-PAGE of cell lysates followed by immunoblotting with polyclonal anti-HPS6 antibodies (HPS6d; 2.5 μg/mL). (A) Band densities of HPS6 at 48 and 72 hours posttransfection with control siRNA (closed bars) or HPS6 siRNA (open bars). Control is shown as 100%. Data represent mean ± SD (n = 3; ***P < .001). (B) Immunoblot of HPS6 (50 μL of lysate from confluent monolayer, ∼0.25 × 106 cells) 72 hours posttransfection with control siRNA (lanes 1, 2, and 3) or HPS6 siRNA (lanes 4, 5, and 6). The membranes were immunoblotted for GAPDH (polyclonal anti-GAPDH, 1 μg/mL) as a loading control. (C) HPS6 knockdown does not affect PDI storage in HUVECs. PDI and HPS6 in HUVECs 72 hours posttransfection with HPS6 or control siRNA were detected by SDS-PAGE of cell lysates, followed by immunoblotting with polyclonal anti-PDI antibody (DL-11; 1 μg/mL) and polyclonal anti-HPS6 antibody (HPS6d; 2.5 μg/mL), respectively. Immunoblot of PDI and HPS6 (25 μL of lysate from confluent monolayer, ∼0.25 × 106 cells) 72 hours posttransfection. HUVEC lysate from cells treated with control siRNA (lanes 1, 2, and 3) or HPS6 siRNA (lanes 4, 5, and 6). Recombinant PDI (15 ng) was run as positive control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
