Fig. 7: Inhibition of BSA-induced HSPA1L secretion by rhVaspin.

a HK2 cells were cultured with BSA 0 or 10 mg/ml at the presence of MG132 0 or 5 μM for 10 hr. BSA-induced decrease of HSPA1L protein levels was partially reversed by MG132. b HEK293T cells expressing p3xFLAG-GFP-HSPA1L were cultured with BSA at 0 or 10 mg/ml. Supernatant of HEK293T cell were immunoprecipitated with ANTI-FLAG M2 affinity agarose gel. Secreted HSPA1L was detected by Western blot analysis using anti-HSPA1L antibody. c HEK293T cells expressing p3xFLAG-GFP-HSPA1L were cultured with BSA at a dose of 0 or 10 mg/ml for 24 h. In addition to BSA, rhVaspin was administrated at a dose of 0, 1, 20, 100 ng/ml. Supernatant of HEK293T cell were immunoprecipitated with ANTI-FLAG M2 affinity agarose gel. Secreted HSPA1L was detected by Western blot analysis using anti-HSPA1L antibody. d Whole cell lysates were subjected to Western blot analyses. Blotting was performed by HSPA1L and normalized by αTubulin. e HK2 cells expressing p3xFLAG-GFP-HSPA1L were cultured with BSA at a dose of 0 or 10 mg/ml for 24 hr. rhVaspin was co-cultured at a dose of 100 ng/ml. Supernatants of HK2 cell were immunoprecipitated with ANTI-FLAG M2 affinity agarose gel. Secreted HSPA1L was detected by Western blot analysis using anti-HSPA1L antibody. f Whole cell lysates were subjected to Western blot analyses. Blotting was performed by HSPA1L and total densitometry quantification of HSPA1L and 3xFLAG-GFP-HSPA1L are normalized by αTubulin. Right panel shows statistical analysis. Data are shown as means  ± S.E. N = 4 (a), 3 (b), 5 (c), 4 (d), 3 (e), 4 and (f), *p < 0.05, **p < 0.01. Western blots were repeated twice.