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. Author manuscript; available in PMC: 2013 Sep 1.

Published in final edited form as: FEBS J. 2012 Jul 16;279(17):3048–3070. doi: 10.1111/j.1742-4658.2012.08686.x

Fig. 6 — (A) Sumo1 and Senp-1-dependent regulation of endogenous LEDGF activity in regulating Hsp27 protein expression. Cells were transfected with different concentrations of pEGFP-Sumo1 (0.5, 1.0 and 2.0 μg). Cell extracts isolated after 48h of transfection were resolved on 10% SDS-PAGE, and Western analysis was conducted using specific antibodies as indicated. The same membrane was utilized to visualize the relative expression levels. (B) Cells were transfected with pFlag-Senp-1 at different concentrations as indicated. Western analysis was carried out and membranes were striped/restriped and immune-stained with Senp-1 or Hsp27 antibodies. β-actin antibody was used as an internal control and to normalize expression. (* p<0.05; **p<0.001)

(C) Disruption of LEDGF Sumoylation motif, K364 (K to R) promoted its transcriptional capacity. Cells were transfected either with pEGFP-vector, pEGFP-LEDGF or pEGFP-K364R and αB crystallin (left part), or Hsp27 (right part) promoters linked to CAT reporter vector. After 72h cell lysates were analyzed for CAT activity. (D) LEDGF siRNA assay showing the involvement of Sumo1 and Senp-1 in modulating transcriptional activity of LEDGF. Cells were transiently cotransfected with pCAT-Hsp27 reporter plasmid and pEGFP-Sumo1 (2 μg) or pFlag-Senp-1 (0.15 μg) or with or without siRNA specific to LEDGF or with empty vector as indicated. CAT activity was monitored. Results are represented as a histogram, and promoter activity was compared between siRNA-LEDGF transfected and untransfected cells (D; left half, black bar and right half, black bar). The data represent the mean ± SD from three independent experiments. (* p<0.05; **p<0.001) statistically significant difference. (E) Silencing of LEDGF by specific siRNA was confirmed through western analysis (upper panel). The membrane was striped and stained with β-actin antibody to normalize expression.

Fig. 6 — (A) Sumo1 and Senp-1-dependent regulation of endogenous LEDGF activity in regulating Hsp27 protein expression. Cells were transfected with different concentrations of pEGFP-Sumo1 (0.5, 1.0 and 2.0 μg). Cell extracts isolated after 48h of transfection were resolved on 10% SDS-PAGE, and Western analysis was conducted using specific antibodies as indicated. The same membrane was utilized to visualize the relative expression levels. (B) Cells were transfected with pFlag-Senp-1 at different concentrations as indicated. Western analysis was carried out and membranes were striped/restriped and immune-stained with Senp-1 or Hsp27 antibodies. β-actin antibody was used as an internal control and to normalize expression. (* p<0.05; **p<0.001)

(C) Disruption of LEDGF Sumoylation motif, K364 (K to R) promoted its transcriptional capacity. Cells were transfected either with pEGFP-vector, pEGFP-LEDGF or pEGFP-K364R and αB crystallin (left part), or Hsp27 (right part) promoters linked to CAT reporter vector. After 72h cell lysates were analyzed for CAT activity. (D) LEDGF siRNA assay showing the involvement of Sumo1 and Senp-1 in modulating transcriptional activity of LEDGF. Cells were transiently cotransfected with pCAT-Hsp27 reporter plasmid and pEGFP-Sumo1 (2 μg) or pFlag-Senp-1 (0.15 μg) or with or without siRNA specific to LEDGF or with empty vector as indicated. CAT activity was monitored. Results are represented as a histogram, and promoter activity was compared between siRNA-LEDGF transfected and untransfected cells (D; left half, black bar and right half, black bar). The data represent the mean ± SD from three independent experiments. (* p<0.05; **p<0.001) statistically significant difference. (E) Silencing of LEDGF by specific siRNA was confirmed through western analysis (upper panel). The membrane was striped and stained with β-actin antibody to normalize expression.