rabbit anti csk  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Zfand5 terminates TLR3/4 signaling and necroptosis by targeting TRIF to the proteasome for degradation

doi: 10.1038/s41419-026-08946-0

Figure Lengend Snippet: HEK293T cells were co-transfected with IFNβ-Luc ( a ) or NF-κB-Luc ( b ) reporter constructs along with Flag-tagged Zfand5 and Myc-tagged TRIF, TBK1, or MyD88 as indicated. Cells were harvested after 36 hours, and luciferase activity was analyzed using the dual-luciferase reporter assay. The expression of transfected proteins in cell lysates was confirmed by immunoblotting (lower panels). c Schematic diagram illustrating the proposed role of Zfand5 in TLR3/4-mediated signaling. d BMDMs were stimulated with LPS (100 ng/ml), poly(I:C) (30 μg/ml), or Pam 3 CSK 4 (100 ng/ml) for the indicated times. e Zfand5 +/+ and Zfand5 –/– RAW 264.7 cells were treated with LPS (100 ng/ml) for 4 h. The protein levels of TRIF, MyD88, Zfand5, and tubulin in cell lysates were analyzed by immunoblotting. Band intensities are presented as fold changes relative to untreated control cells after normalization to Tubulin expression, and are shown below. Zfand5 +/+ and Zfand5 –/– BMDMs were pretreated with cycloheximide (CHX, 2 μg/ml) for 30 mins and then stimulated with LPS (100 ng/ml) ( f ) or poly(I:C) (30 μg/ml) ( g ) for the indicated times. Cell lysates were analyzed by immunoblotting using the indicated antibodies. TRIF and MyD88 band intensities are normalized to GAPDH or tubulin expression and compared to untreated cells. Quantification of three independent experiments is shown in the right or bottom panels. h, i Wild-type (WT) and Zfand5 –/– RAW 264.7 cells or iMEFs were pretreated with CHX (2 μg/ml) for 30 mins and stimulated with LPS (100 ng/ml) for the indicated times. TRIF levels were analyzed as in ( f, g ). j WT and Zfand5 –/– BMDMs or RAW 264.7 cells were treated with LPS (100 ng/ml) or poly(I:C) (30 μg/ml) for the indicated times. The expression levels of the indicated mRNA were analyzed by RT-qPCR. k–p Zfand5 –/– RAW 264.7 cells were reconstituted with a Tet-inducible vector or Flag-tagged Zfand5 construct. k–m Reconstituted cells were pretreated with CHX (2 μg/ml) for 30 mins, followed by stimulation with LPS (100 ng/ml) or poly(I:C) (100 μg/ml) for the indicated times. Cells were pretreated with CHX (2 μg/ml) alone or CHX plus MG132 (2 μM) ( n, o ) or NH 4 Cl (2 μM) ( f ) for 30 mins, followed by stimulation with LPS (100 ng/ml) and CHX in the presence or absence of MG132 ( n, o ) or NH 4 Cl ( p ) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies. TRIF band intensities were normalized to GAPDH or tubulin expression and compared with untreated cells. Quantification of three independent experiments is shown in the right panels. Statistical significance was determined using Student’s t test (* P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant). Data are representative of three independent experiments performed in triplicate and are presented as mean ± SD (error bars).

Article Snippet: Poly(I:C) (#27-4732-01) and Pam 3 CSK 4 (#TLRL-PMS) were purchased from GE Healthcare (Little Chalfont, UK) and Invivogen (San Diego, CA, USA), respectively.

Techniques: Transfection, Construct, Luciferase, Activity Assay, Reporter Assay, Expressing, Western Blot, Control, Quantitative RT-PCR, Plasmid Preparation