cell signaling cat 2971  (Cell Signaling Technology Inc)

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting translocator protein protects against myocardial ischemia/reperfusion injury by alleviating mitochondrial dysfunction

doi: 10.3892/etm.2024.12638

Figure Lengend Snippet: TSPO regulates autophagy and mitophagy via related pathways following A/R stimulation. (A) Representative fluorescence images and quantitative analysis of LC3B expression. Scale bar, 20 µm. (B) Western blotting and (C) densitometry analysis of LC3B, Beclin1 and ATG5 expression levels in si-NC and si-TSPO H9c2 cells with or without A/R stimulation. (D) Western blotting and (E) densitometry analysis of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR expression levels. (F) Western blotting and (G) densitometry analysis of P62, PINK1 and Parkin expression levels. (H) Western blotting and (I) densitometry analysis of Mfn2 and Drp1 protein expression levels. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. TSPO, translocator protein; A/R, anoxia/reoxygenation; si, small interfering; NC, negative control; p-, phosporyalated.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h, then incubated with the following primary antibodies overnight at 4˚C: GAPDH (cat. no. HRP-60004; ProteinTech Group, Inc.), cleaved-caspase-3 (cat. no. 9661S; Cell Signaling Technology, Inc.), caspase-3 (cat. no. 66470-1-Ig; ProteinTech Group, Inc.), Bax (cat. no. 2772S; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. 26593-1-AP; ProteinTech Group, Inc.), TSPO (cat. no. ab109497; Abcam), LC3B (cat. no. ab51520; Abcam), ATG5 (cat. no. 12994S; Cell Signaling Technology, Inc.), Beclin 1 (cat. no. 66665-1-Ig; ProteinTech Group, Inc.), P62 (23214S; ProteinTech Group, Inc.), PINK1 (cat. no. 23274-1-AP; ProteinTech Group, Inc.), Parkin (cat. no. 66674-1-Ig; ProteinTech Group, Inc.), Mfn2 (cat. no. 12186-1-AP; ProteinTech Group, Inc.), Drp1 (cat. no. 12957-1-AP; ProteinTech Group, Inc.), ATP5B (cat. no. 17247-1-AP; ProteinTech Group, Inc.), PI3K (cat. no. 4257S; Cell Signaling Technology, Inc.), phosphorylated (p)-PI3K (cat. no. 4228S; Cell Signaling Technology, Inc.), Akt (cat. no. 9272S, Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060S; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983S; Cell Signaling Technology, Inc.), p-mTOR (cat. no. 2971S; Cell Signaling Technology, Inc.), PKR-like ER kinase (PERK; cat. no. ab229912; Abcam), p-PERK (cat. no. 3179S; Cell Signaling Technology, Inc.), ATF6 (cat. no. 24169-1-AP; ProteinTech Group, Inc.) and inositol-requiring enzyme 1 (IRE1; cat. no. bs16696R, BIOSS).

Techniques: Fluorescence, Expressing, Western Blot, Negative Control

Journal: Communications Biology

Article Title: LCRMP-1 is required for spermatogenesis and stabilises spermatid F-actin organization via the PI3K-Akt pathway

doi: 10.1038/s42003-023-04778-2

Figure Lengend Snippet: a Western blotting analysis and quantification of whole-testis lysate for PI3K–Akt–mTOR/GSK3β signaling related to F-actin dynamics in Lcrmp-1 +/+ and Lcrmp-1 −/− mice ( n = 5, mean ± SD, two-tailed Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001). b Western blotting analysis and quantification of fractionated testicular cells (spermatids, spermatocytes, and others) of Lcrmp-1 +/+ and Lcrmp-1 −/− mice. HEF indicates pooled cell fractions 1–8, which exhibit high expression of LCRMP-1, while LEF indicates pooled cell fractions 9–20, which exhibit low expression of LCRMP-1 ( n = 3, mean ± SD, two-tailed Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: These membranes were blocked with 1% nonfat milk or 1% BSA and incubated with primary antibodies overnight at 4 °C (anti-CRMP-1, Cat. #sc-365348, Santa Cruz biotechnology, 1:1000, Cat. #PA5-34768, Thermo Fisher Scientific, 1:1500; anti-LCRMP-1, homemade , 1:10,000; anti-GSK3β, Cat. #9315, Cell Signaling Technology, 1:1000; anti-phosphorylated GSK3β, Cat. #9336, Cell Signaling Technology, 1:1000; anti-GFP, Cat. #2956, Cell Signaling Technology, 1:1000; anti-cleaved caspase 3, Cat. #9661, Cell Signaling Technology, 1:1000; anti-mTOR, Cat. #2972, Cell Signaling Technology, 1:1000; anti-phosphorylated mTOR, Cat. #2971, Cell Signaling Technology, 1:1000; anti-Akt, Cat. #9272, Cell Signaling Technology, 1:1000; anti-phosphorylated Akt, Cat. #9271, Cell Signaling Technology, 1:1000; anti-PI3K, Cat. #4292, Cell Signaling Technology, 1:1000; anti-phosphorylated PI3K, Cat. #4228, Cell Signaling Technology, 1:1000; anti-p70S6K, Cat. #2708, Cell Signaling Technology, 1:1000; anti-phosphorylated p70S6K, Cat. #9234, Cell Signaling Technology, 1:1000; anti-α tubulin, Cat. #11224-1-AP, proteintech, 1:20,000; anti-GAPDH, Cat. #60004-1-Ig, proteintech, 1:20,000; anti-DDX4, Cat. #ab13840, Abcam, 1:2000) followed by incubation with horseradish peroxidase (HRP)-conjugated antibody for an hour at room temperature.

Techniques: Western Blot, Two Tailed Test, Expressing