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. 2017 Apr 3;16(6):1081–1097. doi: 10.1074/mcp.M116.066159

Fig. 8.

Fig. 8.

Transcriptome analysis, protein quantification, and diaphragm muscle localization of SUMO-related enzymes in control and in mechanically ventilated rats without and with drug treatment. The diaphragm muscle SUMO network was altered during CMV treatment and partially restored by BGP-15. Quantitative PCRs performed on SUMOs (A), E1- and E2 SUMO-conjugating enzymes (B and C), SUMO deconjugases transcripts genes (D), STUbL RNF4 (E), and ATR1, TRIM63, and the circadian Per2 (F) transcripts genes were analyzed on the same samples listed in the supplemental Table 1. Fold changes are normalized by GAPDH housekeeping gene and compared with the control diaphragms (ctrl). Mean ± S.D. of indicated fold changes were performed for each sample in triplicate. G, representative Western blotting panel of total myofibrillar proteins from diaphragm sample lysates were probed with specific antibodies to detect some SUMO conjugases (UBC9, PIAS1, PIAS3, and TRAF7), deconjugases (SENP1, SENP2, SENP5, and SENP6) and E3 ubiquitin ligase RNF4. PIAS3 is detected as a double band indicated with a black bracket immediately below the SENP5 bands. H, protein quantifications of the above-described blot panel. The intensities of the analyzed proteins were quantified by densitometry, normalized with the correspondent loading control (GAPDH), and referred to the control signal. Mean ± S.D. of the indicated fold changes were performed for each sample listed in the supplemental Table 1 in triplicate. The internal figure legend indicates the different fillings of the histogram. I, confocal images of diaphragm cryo-cross-sections from control (ctrl) and different duration of CMV (1d, 5d, and 10d) without and with BGP-15 (10d BGP-15) were incubated with depleted muscle protein anti-UBC9 (upper panel), and anti-RNF4 (lower panel) antibodies show the localization and the variation of the UBC9 and RNF4 protein content along muscle fibers. Pictures are representative of four different sections derived from four independent muscle samples within the same group. Scale bars, 50 μm. Nuclei are stained with DAPI.