(A and B) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct. Spearman correlaįIG 4 Antiviral activity of OTOF is independent of Ca 2+. (C) Correlation between relative OTOF expression (the result of OTOF derived from one patient as '1') in PBMCs and viral loads from untreated patients indicated in panel B. Error bars indicate the standard errors of the means. Total RNA was extracted from PBMCs for quantitative PCR to measure OTOF transcripts normalized to GAPDH levels. (B) Relative expression of OTOF in the PBMCs of healthy donors ( n = 16), patients with HIV-1 who did not receive ART ( n = 26), and patients with HIV-1 who received ART ( n = 20 viral loads of <50 copies/mL). The nonsignificantly differentially expressed genes are shown as black points. The significantly differentially expressed genes are plotted in red with upregulated genes on the right side and downregulated genes on the left side. Each point represents one gene, which had detectable expression in both groups (five untreated patients with HIV-1 infection versus five healthy donors). The log 2 fold change difference is presented on the x axis, and the negative log of the false-discovery rate (FDR) is presented on the y axis. Volcano plot of genes differentially expressed in PBMCs isolated from five independent healthy donors and five independent untreated patients with HIV-1 infection. (A) OTOF was induced in the PBMCs of untreated patients with HIV infection. F Relative luciferase (Luc) activity in the culture medium from HEK293T cells transfected with wild-type mįIG 1 IFN-alpha treatment induces OTOF in macrophages. E Relative luciferase (Luc) activity in the culture medium from HEK293T cells transfected with the miR-19b promoter vector (promt) or empty negative vector (empty) together with transfection with c-Jun-siRNA (cJun-si) or scrambled RNA (scRNA). D Western blot analysis of c-Jun and GAPDH proteins. C Relative luciferase activity in the culture medium from HEK293T cells transfected with the miR-19b promoter vector (promt) or empty negative vector (empty) then exposed to H/R or normoxia (Norm). B Relative luciferase (Luc) activity in the culture medium from HEK293T cells transfected with the miR-19b promoter vector (promt) or empty negative vector (empty) together with transfection with c-Jun-expression vector (c-Jun-ov) or control vector (ctrl). Negative control uses empty vector (Empty promoter) (bottom panel). The potential two c-Jun binding sites in the promoter were deleted (MT promoter) (middle panel). This vector uses GLuc (Gaussia Luciferase) as the promoter reporter and SEAP (secreted alkaline phosphatase) as the internal control for signal normalization (top panel). A miR-17-92 promoter region (positions 5786-8494 in accession# NG-032702) was inserted into pEZX-LvPG04 dual-reporter vector (WT promoter). 4 c-Jun enhances miR-17-92 promoter transcription activity. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate ( Product # 32106).įig. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk.

Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System ( Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel ( Product # NP0302BOX), XCell SureLock™ Electrophoresis System ( Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard ( Product # LC5800). A 37 kDa band corresponding to GAPDH was observed across the cell lines tested. The blot was probed with Anti-GAPDH Polyclonal Antibody ( Product # PA1-987, 1:1,000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate ( Product # A27036, 0.25 µg/mL, 1:4000 dilution). Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), PC-3 (Lane 4) and tissue extract (30 µg lysate) of Ms Brain (Lane 5).

Gapdh western blot lean vs obese mice