Gapdh western blot kda8/7/2023 ![]() ![]() Data are plotted as the means +- standard deviations from three triplicates. At 24 h after infection, cells were lysed to measure luciferase reporter activity and for Western blotting to assess the levels of OTOF and GAPDH using specific antibodies. At 24 h after transfection, cells were infected with 100 ng of HIV-1 -.E- (VSV-G) in the presence or absence of thapsigargin at the indicated doses. (C) 293T cells were transfected with a construct encoding FLAG-tagged OTOF or a mock expression construct. Data are plotted as the means +- standard errors of the means from three independent experiments. The inhibition of viral infection was accordingly calculated for OTOF and mock. At 24 h after transfection, cells were infected with 100 ng of HIV-1 -.E- (VSV-G) in the presence or absence of Ca 2+ (A) or Mg 2+ (B) at the indicated doses. (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. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).įIG 1 IFN-alpha treatment induces OTOF in macrophages. 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). ![]()
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