High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction

A, B

Western blot analysis of p97 levels in RAW264.7 (A) or HT1080 (B) cells treated with additional NaCl (+17, 34 and 51 mM) for 12 h.

C

Immunoprecipitation analysis of the interaction between USP33 and p97 in RAW264.7 treated with additional NaCl (+34 mM) for 12 h.

D

Immunoprecipitation analysis of acetylation of Flag‐p97 in HEK293T cells transfected with Flag‐p97 and then treated with additional NaCl (+17, 34 and 51 mM) for 12 h.

E, F

Mass spectrometry analysis of acetylation of Flag‐p97 (E) in RAW264.7 transfected with Flag‐p97 and then treated with additional NaCl (+34 mM) or control ddH₂O (Ctrl) for 12 h. The intensity of p97‐K663 site was shown in (F).

G

Immunoprecipitation analysis of acetylation of Myc‐p97 in HEK293T cells transfected with Myc‐p97 (WT, K663R or K651R) and then treated with additional NaCl (+34 mM) for 12 h.

H

Immunoprecipitation analysis of the interaction between USP33 and Myc‐p97 in HEK293T cells transfected with Myc‐p97 (WT or K663R).

I

Western blot analysis of USP33 levels in HEK293T cells transfected with empty vectors (−) or Myc‐p97 (WT or K663R).

J

Western blot analysis of Flag‐Viperin levels in HEK293T cells co‐transfected with Flag‐Viperin and Myc‐p97 (WT or K663R).

K

Immunoprecipitation analysis of the interaction between USP33 and Myc‐p97 in HEK293T cells transfected with Myc‐p97 (WT or K663R) and then treated with additional NaCl (+34 mM) for 12 h.

L

Western blot analysis of FH‐USP33 levels in p97‐KO HEK293T cells co‐transfected with Myc‐p97 (WT or K663R) and Flag‐USP33, and then treated with additional NaCl (+34 mM) for 12 h.

M

Immunoprecipitation analysis of Viperin ubiquitination in p97‐KO 2fTGH cells co‐transfected with Myc‐p97 (WT or K663R), Flag‐Viperin, and HA‐Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 h.

Figure 6. High salt promotes p97 acetylation to recruit USP33 for degradation.