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. 2021 Nov 15;23(1):e53466. doi: 10.15252/embr.202153466

Figure EV4. High salt downregulates USP33, which is a key deubiquitinase of Viperin.

Figure EV4

  • A
    Immunoprecipitation analysis of ubiquitination of Flag‐Viperin in HEK293T cells co‐transfected with Flag‐Viperin and HA‐Ub, and then treated with MG132 (10 μM) and additional NaCl (+34 mM) for 12 h.
  • B
    Western blot analysis of UBE4A in RAW264.7 cells treated with additional NaCl (+17, 34, and 51 mM) for 12 h.
  • C–E
    Western blot analysis of Flag‐HA (FH)‐USP33 (C), FH‐USP1 (D), FH‐UCHL1 (E) in HEK293T cells transfected with the corresponding plasmids and then treated with additional NaCl (+17 and 34 mM) for 12 h.
  • F
    Immunoprecipitation analysis of the interaction between Myc‐Viperin and HA‐USP33 in HEK293T cells co‐transfected with Myc‐Viperin and HA‐USP33.
  • G
    Western blot analysis of Viperin in 2fTGH cells transfected with increasing amount of FH‐USP33 and then treated with IFNα (5,000 IU/ml) for 15 h.
  • H
    Western blot analysis of Viperin in RAW264.7 transfected with empty vectors (Ctrl) or FH‐USP33 and then treated with mIFNβ (500 and 1,000 IU/ml) for 12 h.
  • I
    Western blot analysis of Viperin in HeLa cells transfected with FH‐USP33 and then treated with IFNα (1,000 IU/ml) for different times.
  • J
    Western blot analysis of USP33 protein levels in two clones (1# and 2#) of Usp33+/+ and Usp33 −/− MEF cells.
  • K
    Western blot analysis of FH‐Viperin in HEK293T cells co‐transfected FH‐Viperin and FH‐USP33 (wild type, WT; C194S and H673Q, DM).
  • L
    Immunoprecipitation analysis of Myc‐Viperin ubiquitination in HEK293T cells co‐transfected with Myc‐Viperin, HA‐Ub, and increasing amount of FH‐USP33.
  • M
    Immunoprecipitation analysis of Flag‐Viperin ubiquitination in HEK293T cells co‐transfected with Flag‐Viperin, HA‐Ub, and shCtrl or shUSP33.
  • N
    Immunoprecipitation analysis of Flag‐Viperin ubiquitination in HEK293T cells co‐transfected with Flag‐Viperin and shCtrl or shUSP33.
  • O
    Western blot analysis of Flag‐Viperin levels in HEK293T cells co‐transfected Flag‐Viperin and FH‐USP33, and then treated with CHX (50 μg/ml) as indicated.
  • P
    Western blot analysis of USP33 in RAW264.7 cells treated with additional NaCl (+17, 34, and 51 mM) for 12 h.
  • Q
    Western blot analysis of USP33 in 2fTGH cells treated with additional NaCl (+34 mM) for different times.
  • R
    Western blot analysis of USP33 in HepG2 cells treated with additional NaCl (+17, 34, and 51 mM) for 12 h.
  • S
    Western blot analysis of Myc‐Viperin in HEK293T cells co‐transfected with Myc‐Viperin and shCtrl (−) or shUSP33, then treated with additional NaCl (+34 mM) for 12 h.
  • T
    Western blot analysis of VSV‐G in Usp33+/+ and Usp33 −/− mouse liver cells infected with VSV (MOI = 1.0, 24 h) immediately after addition of NaCl (+34 mM).

Data information: Data (A–T) are representative of at least two biological replicates.

Source data are available online for this figure.