Journal: Communications Biology

Article Title: The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish

doi: 10.1038/s42003-023-04711-7

Figure Lengend Snippet: a The time gradient experiment of empty vectors or calpain2a-Myc plasmids together with TRAF6-Flag was conducted in EPC cells. The cell lysates were subjected to IB with anti-Myc, anti-Flag, and anti–Tubulin Abs. RNA was extracted from cells and reverse transcribed, then TRAF6 and actin were amplified by PCR primers. b EPC cells were seeded in 12-well plates overnight and co-transfected with TRAF6-Flag and calpain2a-Myc (0.3, 0.6, or 0.9 μg) for 48 h. The expression of TRAF6-Flag and calpain2a-Myc proteins were detected by Western blotting. RNA was extracted from cells and reverse transcribed, then TRAF6 and actin were amplified by PCR primers. c calpain2a-Myc or empty vectors were co-transfected with TRAF6-Flag into EPC cells. At 36 h post-transfection, the transfected cells were treated with cycloheximide (CHX) for 2 or 4 h. d MKC cells were transfected with calpain2a-Flag or empty vectors. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. e , f EPC cells were transfected with the indicated plasmids in the presence or absence of MG132, 3-MA, NH 4 CL, E-64 (50 or 75 μM), or PMSF for 6 h before immunoblot analysis was performed. g MKC cells were transfected with calpain2a-Flag in the presence or absence of E-64 (50 or 75 μM) for 6 h before immunoblot analysis was performed. h calpain2a-Flag and calpain2a-ΔCysPc-Flag were co-transfected with TRAF6-HA into EPC cells. At 48 h post-transfection, the cell lysates were subjected to IB with indicated Abs. i calpain2a-Flag and calpain2a-ΔCysPc-Flag were co-transfected into MKC cells. At 48 h post-transfection, the cell lysates were subjected to IB with anti-TRAF6, anti-Flag, and anti-Tubulin Abs. j IL-1β , IL-8 mRNA in MKC stably transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and treated with saline (0) or challenged with LPS for 4 h ( n = 3 per group). Relative mRNA level was normalized to the expression of the gene encoding β-actin in each sample. All experiments were performed in at least three independent experiments. Data were analyzed by two-way ANOVA ( j ). ** p < 0.01.

Article Snippet: The proteasome inhibitor used at a final concentration: MG132 (20 μM) (Sigma), 3-MA (2 mM) (Sigma), NH 4 Cl (20 mM) (Sigma), E-64 (50 μM) (Solarbio), PMSF (100 μM) (Beyotime), and the cycloheximide (CHX) (Beyotime) used at a final concentration of 100 μg/ml.

Techniques: Amplification, Transfection, Expressing, Western Blot, Stable Transfection, Transduction

Journal: Computational and Structural Biotechnology Journal

Article Title: Phosphorylation of T897 in the dimerization domain of Gemin5 modulates protein interactions and translation regulation

doi: 10.1016/j.csbj.2022.11.018

Figure Lengend Snippet: Mutations T897A and T897E do not affect protein stability. HEK293 cells expressing the wild-type version of G5 845-1508 , side by side with mutants T897A and T897E for 12 h were treated (+) or not (−) with cycloheximide (CHX) for additional 16 h. Samples were taken at 0, 12, and 16 h post–CHX treatment. The intensity of each protein at the indicated time was determined by WB. Bars represent the protein intensity (mean ± SEM) of three independent experiments relative to time 0 in each case. The differences between values were not significant in all cases.

Article Snippet: For cycloheximide (CHX) chase experiments, CHX (100 μg/ml) (Merck) was added to stop translation at 12 h post-transfection.

Techniques: Expressing