Figure 4. Enhanced ATF4 mRNA translation in PTP1B-deficient differentiated brown adipocytes.

Differentiated WT (A, B) and PTP1B KO (C, D) brown adipocytes were left untreated (A, C) or treated (B, D) with thapsigargin (TG, 1 µM) for 2 hr followed by polysome profile analysis using 20–50% sucrose gradients as described in Methods. Gradients were fractionated from top (fraction 1) to bottom (fraction 17). Absorbance of the gradients was measured continuously at 264 nm to provide the polysome profiles. Association of ATF4 mRNA and GAPDH mRNA with the ribosomal interface was detected by RT-PCR. Data present one of two reproducible experiments. E) Protein synthesis in differentiated WT, KO and D/A brown adipocytes before and after TG treatment. Bar chart represents newly incorporated ³⁵S methionine from three independent experiments. (*) indicates significant difference between KO and D/A versus WT at the corresponding treatment time and (^#) indicates significant difference between treated and non-treated cells within each group.