Fig 1: KHSRP binding to specific mRNAs is inhibited by oxygen and glucose deprivation. HEK293T cells were exposed to 24 hours of normoxia or hypoxia (1% O2) in the presence of 1 g/L glucose for 24 hours and then subjected to KHSRP immunoprecipitation. (A) Fold enrichments calculated from immunoprecipitated mRNA levels normalizing to GAPDH as a negative control and the normal rabbit serum negative control IP. (B) Representative Western blot showing equivalent amounts of KHSRP protein being immunoprecipitated (IP) under Normoxic (N) or Hypoxic (H) conditions, antibody heavy chain (Ab HC) was also detected. (C) Representative western blot showing extent of KHSRP protein knockdown in normoxic conditions after 72 hours siRNA treatment as compared with negative control siRNA (Neg). GAPDH protein levels were used as a loading control. (D) HEK293T cells were transfected with siRNA targeting KHSRP or negative control siRNA (Neg) for 48 hours before being exposed to 24 hours of Normoxia or Hypoxia (1% O2) in the presence of 1 g/L glucose. Cells were treated with Actinomycin D for 0, 1 or 2 hours, mRNA levels of VEFG165 and MYC were determined via qPCR, normalized to GAPDH and 0 hour time point and half-lives determined. Data represents average of N = 3 ± SD for each condition. *P ≤ 0.05 from respective Normoxic condition.