Figure 6.
PTP1BWT activity and stability remains unaltered upon co-expression of PTP1B∆2–4. (A) Phosphatase assay with Strep-tagged PTP1BWT alone or together with HA-PTP1BWT, HA-PTP1BC215S, HA-PTP1B∆6 or HA-PTP1B∆2–4 ectopically expressed in HEK293 cells, as indicated. pEXPR-IBA105 (EV) serves as control. Mean values of three independent experiments and standard error of mean (SEM) are depicted. Comparisons were performed between PTP1BWT measurement and individual transfections. Significances are calculated using Student’s t-test (P < 0.05 was regarded as significant). (B) Immunoblot analysis of whole cell extracts from HEK293 cells either transfected with pcDNA3.1 (EV), or with Strep-PTP1BWT and increasing amounts of HA-PTP1B∆6 (1:50, 1:5, 1:2, 1:1). Antibodies against PTP1B and β-actin used to determine amounts of PTP1BWT (endogen and exogen), PTP1B∆6 and the housekeeper β-actin. (C) Immunoblot analysis of whole cell extracts from HEK293 cells either transfected with pcDNA3.1 (EV), with Strep-PTP1BWT or with Strep-PTP1BWT and increasing amounts of HA-PTP1BC215S or HA-PTP1B∆2–4 (1:50, 1:5, 1:2, 1:1). Antibodies against Strep-tag used to determine amounts of exogen PTP1BWT, against HA-tag to determine amounts of PTP1BC215S or HA-PTP1B∆2–4, antibodies against PTP1B used to determine amounts of whole PTP1B and antibodies against the housekeeper β-actin. (D) Relative mRNA expression of the PTPN1WT RNA levels expressed in HEK293 cells with or without increasing amounts of PTP1B∆6 (1:50, 1:5, 1:2, 1:1) measured by sqPCR. Values normalized against the mRNA expression levels of the two housekeeper genes β-actin and GAPDH and calculated in comparison to the ectopic PTP1BWT expression alone. Mean and SEM of three independent experiments depicted. (E) Quantification of the amount of PTP1BWT protein as depicted in (B). Mean value and SEM of six independent experiments is shown and significance was determined in comparison to the PTP1BWT alone measurements. (F) Quantification of the amount of PTP1BWT protein as depicted in (C). Mean value and SEM of three independent experiments is shown and significances are calculated using Student’s t-test determined in comparison to the PTP1BWT alone measurement (P < 0.05 was regarded as significant). (G) Strep-tag immunoprecipitation (IP, upper 4 blots) using WCE of HEK293 cells transfected with Strep-tagged PTP1BWT alone or together with HA-PTP1BWT, HA-PTP1B∆6 or HA-PTP1B∆2–4. HEK293 cells transfected with Strep-tagged NEMO alone and in combination with untagged IKK2 served as IP positive control. Immunoblots (IB, lower 2 blots) using the same WCE served as intake control. Protein bands visualized using indicated antibodies.