Supplemental Figures

This article contains the following supporting material:

• Figure S2 - Exogenously-expressed UBPY and UBPY^ΔSBM are accumulated on endosomes in cells overexpressing Hrs or SKD1^E235Q

  (A-A”, B-B”) HeLa cells were transfected with FLAG-tagged UBPY (A-A”) or UBPY^ΔSBM (B-B”) together with HA-tagged Hrs, and double-stained with anti-HA (A, B) and anti-FLAG (A’, B’) antibodies. Asterisks indicate cells expressing FLAG-UBPY (A’, A”) or FLAG-UBPY^ΔSBM (B’, B”) but not HA-Hrs. (C-C”, D-D”) HeLa cells were transfected with HA-tagged UBPY (C-C”) or UBPY^ΔSBM (D-D”) together with GFP-tagged SKD1^E235Q, and stained with anti-HA antibody (C’, D’). C and D show the localization of SKD1E235Q detected by the GFP fluorescence. A”, B”, C”, and D” are merged images. Arrowheads indicate co-localization of UBPY and UBPY^ΔSBM with transfected Hrs (A-A”, B-B”) and SKD1^E235Q (C-C”, D-D”). N indicates the nucleus. Bar, 20 μm.

• Figure S1 - UBPY is not stably associated with the Hrs-STAM complex

  (A) Lysates of HeLa cells stimulated with EGF for indicated periods were immunoprecipitated with anti-STAM1 antibody. The immunoprecipitates as well as the total cell lysates were immunoblotted with anti-UBPY (top panel), anti-Hrs (middle panel), and anti-STAM1 (bottom panel) antibodies. Asterisks in the top panel indicate non-specific bands as they were also detected in UBPY-depleted cells. (B) Lysates of HeLa cells stimulated with EGF for indicated periods were immunoprecipitated with anti-UBPY antibody. The immunoprecipitates as well as the total cell lysates were immunoblotted with anti-STAM1 (top panel) and anti-UBPY (bottom panel) antibodies.

• Figure S3 - UBPY^C748A localizes on endosomes in Hrs-depleted cells

  HeLa cells were co-transfected with HA-UBPY^C748A and Hrs siRNA expression vectors, and stained with anti-HA antibody (A, B) together with anti-EEA1 (A’) or anti-LAMP1 (B’) antibody. A” and B” are merged images. Arrowheads indicate co-localization of UBPY^C748A with EEA1 (A-A”). N indicates the nucleus. Bar, 20 μm.