Fig 1: Sigma-1 receptor transcription, protein expression, and surface level do not change after SNI in rat DRGs. a Sample blots of sigma-1 receptor (Sig-1R) expression at different time points after SNI. a-Tubulin served as internal control. b Summary data show that Sig-1R protein expression decreases slightly at day 14 (nonsignificant per ANOVA) but not at days 3 or 28 after SNI (left panel). The Sig-1R mRNA does not change after SNI at days 3, 14, or 28 (right panel). c Surface level of Sig-1Rs remains unchanged after SNI. Streptavidin beads were used to pull down biotinylated lysate (200 µg) from DRGs 14 days after SNI or sham surgery. Na+-K+ ATPase served as internal control and GAPDH served as negative control. d Sig-1R KO increases Cav2.2 expression in HEK cells. Western blotting shows an increase of Cav2.2 protein level in total cellular lysates of Sig-1R KO HEK cells when compared to wild-type cells. Data represent means ± SEM; two-way ANOVA or Student’s t test
Fig 2: Sig-1R regulates 4E-BP1 and its effect on eIF4E. a Sig-1R KO decreases the transcription and translation of 4E-BP1 in HEK cells. Sample blot demonstrates a decrease of 4E-BP1 protein in Sig-1R KO HEK cells. Summary data reveal an elevated level of protein and mRNA of 4E-BP1 in Sig-1R KO HEK cells. b Sig-1R knockout (KO) increases eIF4E and Cav2.2 mRNA binding. In the RNA-IP test, eIF4E binding to Cav2.2 5'cap region increases in Sig-1R KO HEK cells. Data represent means ± SEM; Student’s t test; Mann-Whitney test for b; *P < 0.05, ***P < 0.001
Fig 3: Exogenous expression of Sigma-1-Dsred receptor and Cav2.2-GFP in HEK-293T cell line. (A) Fluorescence images of HEK-293T cells transfected with 0.5 µg Sigma-1-Dsred receptor cDNA. (B) Fluorescence images of HEK-293T cells transfected with 0.5 µg Cav2.2-GFP (a1b+ß1b+a2d1subunits) cDNA. (C) Confocal microscopy images of HEK-293T cells with both 0.5 µg Cav2.2-GFP and 0.5 µg Sigma-1-Dsred receptor cDNA. Co-localization was shown in yellow.
Fig 4: Cav2.2 protein expression is downregulated in DRGs after SNI. a Total Cav2.2 protein expression deceases after SNI. Sample bands of Cav2.2 expression were demonstrated at different time after sham surgery or SNI from rat DRGs. a-Tubulin served as internal control. Summary data showed that Cav2.2 protein expression but not mRNA level decreases after SNI on day 3, 14, or 28. b Cav2.2 protein level decreases at the plasma membrane after SNI. Cav2.2 at plasma membrane decreases after day 14 of SNI. N-Cadherin expression from streptavidin beads pulled down served as internal control. Data are means ± SEM; two-way ANOVA, or Student’s t test; *P < 0.05, **P < 0.01
Fig 5: cFOS increases 4E-BP1 transcription via its interaction with Sig-1R. a Overexpression of 4E-BP1 decreases Cav2.2 in HEK cells. b Sig-1R interacts with cFOS in HEK cells. In cFOS-Myc/DDK and Sig-1R-GFP HEK co-expressing HEK cells, sample blot demonstrates the co-immunoprecipitation of those two proteins (lane 6). c Co-overexpressed Sig-1R and cFOS bind to the promoter of 4E-BP1. DNAs obtained from chromatin immunoprecipitation assays testing the binding of GFP-tagged Sig-1R (upper panel) or Myc-tagged cFOS (lower panel) to the 4E-BP1 promoter in HEK cells were examined. The precipitated DNA was amplified using specific primers for the 4E-BP1 promoter. Quantified results from the PCR products were normalized to input. Summary data are presented as means ± SEM; Student’s t test; *P < 0.05, ***P < 0.001; N = 3
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