anti cyld rabbit  (Proteintech)

Journal: Advanced Science

Article Title: Cleavage‐Resistant CYLD Protects Against Autoimmune Hepatitis

doi: 10.1002/advs.202513015

Figure Lengend Snippet: CYLD inhibits MEK1/2 activation by removing K63‐linked ubiquitin chains. (A,B) Immunoblot analysis of MAPK and NF‐κB signaling in PMs of the indicated genotypes after stimulation with S100A9 (400 ng/mL). (C–F) HEK293T cells were transfected with the indicated plasmids or corresponding empty vector for 24 h, followed by immunoprecipitation with anti‐Flag beads and immunoblot analysis. (G) Domain architecture of full‐length CYLD and truncation mutants. (H) HEK293T cells were transfected with Flag‐MEK1 and HA‐CYLD (or truncation mutants) for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (I,J) HEK293T cells were transfected with Flag‐MEK1, Myc‐CYLD (or catalytic mutants C600A/S), HA‐Ub‐WT, and the corresponding empty vector as indicated for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (K) HEK293T cells stably expressing control or Cyld single‐guide (sg) RNA were transfected with Flag‐MEK1, HA‐Ub‐WT, and empty vector as indicated for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (L,M) HEK293T cells were transfected with Flag‐MEK1, Myc‐CYLD (or catalytic mutants C600A/S), HA‐Ub‐K63, and corresponding empty vectors as indicated for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (N) HEK293T cells stably expressing control or Cyld single‐guide RNA were transfected with Flag‐MEK1, HA‐Ub‐K63, and empty vectors as indicated for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies.

Article Snippet: The following antibodies were used for western blotting and immunoprecipitation experiments: CYLD (1:1000, Cell Signaling Technology, 8462), GAPDH (1:20 000, Sigma, G9545), P65 (1:1000, Cell Signaling Technology, 8242), p‐P65 (1:1000, Cell Signaling Technology, 3033), IκBα (1:2000, Cell Signaling Technology, 9242), p‐IκBα (1:1000, Cell Signaling Technology, 9246), MEK1/2 (1:1000, Cell Signaling Technology, 9122), p‐MEK1/2 (1:1000, Cell Signaling Technology, 9121), ERK1/2 (1:1000, Cell Signaling Technology, 9102), p‐ERK1/2 (1:1000, Cell Signaling Technology, 9101), P38 (1:1000, Cell Signaling Technology, 9228), p‐P38 (1:1000, Cell Signaling Technology, 9211), JNK (1:1000, Cell Signaling Technology, 9252), p‐JNK (1:1000, Cell Signaling Technology, 9251), HA‐Tag (1:3000, Cell Signaling Technology, 3724), Myc‐Tag (1:2000, Cell Signaling Technology, 2276), Flag‐Tag (1:20 000, Sigma, A8592‐1MG), K63‐linkage specific polyubiquitin (1:1000, Cell Signaling Technology, 5621).

Techniques: Activation Assay, Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing, Control

Journal: Advanced Science

Article Title: Cleavage‐Resistant CYLD Protects Against Autoimmune Hepatitis

doi: 10.1002/advs.202513015

Figure Lengend Snippet: TRIM25 cooperates with CYLD to regulate MEK1/2 ubiquitination and activation. (A,B) HEK293T cells were transfected with Myc‐TRIM25 and Flag‐MEK1 (or Flag‐MEK2) with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (C) Domain architecture of full‐length TRIM25 and truncation mutants. (D) HEK293T cells were transfected with HA‐MEK1 and Flag‐TRIM25 (or truncation mutants) for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (E,F) HeLa cells were transfected with HA‐Ub‐WT, Myc‐TRIM25, and Flag‐MEK1/2 (or mutants) with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (G) HEK293T cells were transfected with HA‐Ub‐WT, Myc‐CYLD, and Flag‐MEK1 (or mutants) with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (H,I) HEK293T cells were transfected with Myc‐TRIM25 and Flag‐MEK1/2 (or mutants) with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (J) HEK293T cells were transfected with Flag‐MEK1, Myc‐RAF1, and HA‐Ub‐WT with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (K,L) HEK293T cells were transfected with HA‐RAF1 and Flag‐MEK1/2 (or mutants) with the indicated combinations for 24 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag beads and immunoblotted with the indicated antibodies. (M,N) WT mice were pretreated with trametinib (5 mg/kg, i.p.) or vehicle 2 h before ConA challenge (12 mg/kg, i.v.). Serum levels of ALT, AST, IL‐6, and TNFα were measured at 12 h post‐injection (n = 8 mice per group). (M) AST and ALT. (N) IL‐6 and TNFα. Statistical analysis for panels M and N was performed using a two‐tailed unpaired Student's t ‐test. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used for western blotting and immunoprecipitation experiments: CYLD (1:1000, Cell Signaling Technology, 8462), GAPDH (1:20 000, Sigma, G9545), P65 (1:1000, Cell Signaling Technology, 8242), p‐P65 (1:1000, Cell Signaling Technology, 3033), IκBα (1:2000, Cell Signaling Technology, 9242), p‐IκBα (1:1000, Cell Signaling Technology, 9246), MEK1/2 (1:1000, Cell Signaling Technology, 9122), p‐MEK1/2 (1:1000, Cell Signaling Technology, 9121), ERK1/2 (1:1000, Cell Signaling Technology, 9102), p‐ERK1/2 (1:1000, Cell Signaling Technology, 9101), P38 (1:1000, Cell Signaling Technology, 9228), p‐P38 (1:1000, Cell Signaling Technology, 9211), JNK (1:1000, Cell Signaling Technology, 9252), p‐JNK (1:1000, Cell Signaling Technology, 9251), HA‐Tag (1:3000, Cell Signaling Technology, 3724), Myc‐Tag (1:2000, Cell Signaling Technology, 2276), Flag‐Tag (1:20 000, Sigma, A8592‐1MG), K63‐linkage specific polyubiquitin (1:1000, Cell Signaling Technology, 5621).

Techniques: Ubiquitin Proteomics, Activation Assay, Transfection, Immunoprecipitation, Injection, Two Tailed Test