Fig. 2: TSPAN4 is ubiquitinated for degradation.
From: O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release
a HeLa cells stably expressing TSPAN4-Flag were treated with 100 μM CHX for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to GAPDH of three independent experiments was quantified. Error bars, mean ± SD. Source data are provided as a Source Data file. b HeLa cells stably expressing TSPAN4-Flag were treated with 100 μM CHX for 1 h, and further treated with 100 μM CQ or 10 μM MG132 for 6 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to GAPDH of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. c, d HeLa cells stably expressing TSPAN4-Flag were infected with or without VSV-GFP or HSV-1 at an MOI of 1 for indicated time, and were further treated with or without 10 μM MG132 or 100 μM CQ for 6 h. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to Tubulin of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file. e HEK293T cells were transfected with HA-UbK48 with or without TSPAN4-Flag for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. f HEK293T cells were transfected with HA-UbK48 and TSPAN4-Flag mutants for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. g Amino acid sequence from 106 to 238 of TSPAN4 and point mutations used in this study. h 6 K (K110, K120, K121, K187, K195, K232) are the main ubiquitin site of TSPAN4. HEK293T cells were transfected with HA-UbK48 and TSPAN4-Flag or its mutants for 36 h. Interactions were analyzed by co-immunoprecipitation (n = 3 independent experiments). Source data are provided as a Source Data file. i HeLa cells were transfected with TSPAN4-Flag or TSPAN46KR-Flag for 24 h, and cells were further treated with 100 μM CHX for indicated time. Lysates were analyzed via WB. Relative intensity of TSPAN4 normalized to Tubulin of three independent experiments was quantified. Error bars, mean ± SD. Two-tailed Unpaired Student’s t-test. Source data are provided as a Source Data file.
