IL-6 regulates Bcl-2 family expression and binding to Bim. MM.1s (A) and KMS18 (B) cells were cocultured with Hs-5 cells, 50% conditioned media from Hs-5 cells, or treated with 10 ng/mL IL-6 for 24 hours. Lysates were prepared and subject to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting for Bim, Mcl-1, Bcl-x_L, Bcl-2, and actin. (C) The 8226, KMS18, MM.1s, OPM2, and U266 cells were treated with 10 ng/mL IL-6 for 24 hours. Cells were then lysed for protein and western blotting or RNA and quantitative reverse transcription PCR (qRT-PCR). A representative western blot of 3 independent experiments is shown. (D) Changes in protein expression were quantitated by densitometry after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. (E) RNA expression of Mcl-1 and Bcl-2 were measured using qRT-PCR. Both graphs depict the mean percent change ± SE relative to untreated cells (*P < .05 by the 1 sample Student t test). (F) MM.1s and KMS18 were treated with 1 ng/mL IL-6 for 24 hours, and protein lysates were prepared then subjected to coimmunoprecipitation with anti-Mcl-1, anti-Bcl-x_L, and anti-Bcl-2 antibodies. The resulting protein complexes and protein input (WCL) were examined by western blot analysis using anti-Bim, anti-Mcl-1, anti-Bcl-x_L, and anti-Bcl-2. The proportion of Bim bound to Mcl-1, Bcl-x_L, and Bcl-2 was quantitated by densitometry and is represented as a pie chart for each condition. Each pie chart is the average of 3 independent experiments.