PTBP1 expression changes across the cell cycle. (A) Chemical inhibitors used to arrest cells at specific phases of the cell cycle: thymidine, arrest at G1/S, and RO3306 or Nocodazole, arrest at G2/M transitions. Cells were synchronized and collected at time points after release from the drugs. (B) Representative western blot of whole cell lysates of synchronized HEK293T cells prepared using synchronized samples. Separate methods to arrest G2/M were used. M-a and M-b samples were synchronized with the use of RO3306 and Nocodazole, respectively. PTBP1-2 and PTBP1-4 protein isoforms have similar sizes and comigrate in the gel. Below the gel are shown the amounts of the PTBP1 isoforms relative to that in G1/S phase, normalized to HSP90 levels. (C) Amounts of total PTBP1 mRNA were assessed using quantitative PCR for each phase of the cell cycle. ΔCT values normalized to ACTB mRNA levels. ns, not statistically significant; P-values >0.2. Experiments were carried out in biological triplicate, with standard deviations shown. Async, proliferating cells without synchronization.