Figure 1. Knockdown of CIB1 and CIB2 in Jurkat T-cells.

Jurkat cells were transduced by lentiviral vectors to express shRNAs targeting CIB1 or CIB2, and carrying a selectable marker (puromycin). After expansion and selection of shRNA-transduced cells, each population was evaluated for their knockdown efficiency and viability before challenge with HIV-1. (A) Structure of mRNA transcripts for CIB1 and CIB2 and shRNA target sites. Total RNA was extracted and cDNA was synthesized for quantification of CIB1, CIB2 and GAPDH mRNA levels. The ratio of CIB1/GAPDH mRNA (B) and CIB2/GAPDH mRNA (C) are expressed relative to those of non-transduced (NT) Jurkat cells. Knockdown at the protein level was confirmed by Western-blotting using antibodies recognizing CIB1 (clone 791119, R&D systems) (D), CIB2 (clone CIB2C12B11, Abcam) (E) and GAPDH (clone 6C5, Santa Cruz Biotechnology). Gels were run under the same experimental conditions and cropped images are shown (full-length blots are presented in Supplementary Figure S2). (F) After expansion and selection, the viability of shRNA-transduced cells was measured by Trypan blue exclusion. For panels B, C and F, values represent mean ± SEM of 4 or more independent transductions. *P < 0.05, **P < 0.01 versus sh-SCRAM (Wilcoxon paired test).