Figure S2. PTPN23 is required for... | Rockefeller University Press

Figure S2.

$PTPN23 is required for PI3KC2α-mediated production of PI(3,4)P2 and AKT activation. (A) DiFMUP assay detecting phosphatase activity of the PTPN23 CD (Ile1211 to Val1450), along with C1392S, S1394A, and C1392S-S1394A mutants (n = 3). Two-way ANOVA, Tukey’s test. (B) Immunoblot showing protein levels of PTPN23 and Rab5 after cell fractionation in A375 and SK-MEL-28 cells. (C) Immunofluorescence showing the subcellular localization of PTPN23 and Rab5 in A375 and SK-MEL-28 cells. Scale bar, 20 μm. (D) Pathway enrichment analysis of PTPN23-interacting proteins identified by LC-MS/MS. (E) Peptide counts of PTPN23 and PI3KC2α from affinity mass spectrometry. (F) Immunofluorescence showing the subcellular localization of PTPN23 and PI3KC2α in A375 and SK-MEL-28 cells. Scale bar, 20 μm. (G) Immunoblot analysis of PTPN23 and PI3KC2α after cell fractionation in A375 and SK-MEL-28 cells. (H) Immunoblot showing total and phosphorylated proteins in cell fractionation from A375 cells after PTPN23 depletion. (I) Immunofluorescence and quantification of PI(3,4)P2 levels after PTPN23 depletion in A375 cells with or without exogenous soluble PI(3,4)P2 treatment (n = 3). Scale bar, 20 μm. One-way ANOVA, Tukey’s test. ****P < 0.0001; ***P < 0.001; *P < 0.05. WCL: whole-cell lysates; HM: heavy membrane; Endo: endosome. Source data are available for this figure: SourceData FS2. Refer to the image caption for details.$

PTPN23 is required for PI3KC2α-mediated production of PI(3,4)P2 and AKT activation. (A) DiFMUP assay detecting phosphatase activity of the PTPN23 CD (Ile1211 to Val1450), along with C1392S, S1394A, and C1392S-S1394A mutants (n = 3). Two-way ANOVA, Tukey’s test. (B) Immunoblot showing protein levels of PTPN23 and Rab5 after cell fractionation in A375 and SK-MEL-28 cells. (C) Immunofluorescence showing the subcellular localization of PTPN23 and Rab5 in A375 and SK-MEL-28 cells. Scale bar, 20 μm. (D) Pathway enrichment analysis of PTPN23-interacting proteins identified by LC-MS/MS. (E) Peptide counts of PTPN23 and PI3KC2α from affinity mass spectrometry. (F) Immunofluorescence showing the subcellular localization of PTPN23 and PI3KC2α in A375 and SK-MEL-28 cells. Scale bar, 20 μm. (G) Immunoblot analysis of PTPN23 and PI3KC2α after cell fractionation in A375 and SK-MEL-28 cells. (H) Immunoblot showing total and phosphorylated proteins in cell fractionation from A375 cells after PTPN23 depletion. (I) Immunofluorescence and quantification of PI(3,4)P2 levels after PTPN23 depletion in A375 cells with or without exogenous soluble PI(3,4)P2 treatment (n = 3). Scale bar, 20 μm. One-way ANOVA, Tukey’s test. ****P < 0.0001; ***P < 0.001; *P < 0.05. WCL: whole-cell lysates; HM: heavy membrane; Endo: endosome. Source data are available for this figure: SourceData FS2.