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: Cellular and molecular gastroenterology and hepatology

Article Title: Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

doi: 10.1016/j.jcmgh.2022.12.008

Figure Lengend Snippet: Figure 4. Smo deletion promote lipogenesis by activating mTOR. (A) Hepatic free fatty acids (pmol/mg) in control and Smo- KO mice. (B) Protein immunoblots of SREBP1c and peroxisome proliferator-activated receptor (PPARg1) in control and Smo- KO mice treated with saline or insulin. (C, D) Protein immunoblots of pmTOR Ser 2448 and its downstream effectors pS6K1 and p4E-BP1 in control and Smo-KO mice. (E) Protein immunoblots of pmTOR Ser 2481 in control and Smo-KO mice. The error bar indicates SEM. *P .05; ** P :01:

Article Snippet: Table 3.List of Antibodies for Western Blotting and IHC Primary Antibody Supplier Reference Dilution b-tubulin Abcam Cat. #ab6046 1:3000 SREBP2 Abcam Cat. #ab30682 1:1000 pAkt Ser 473 Cell Signaling Cat. #4060 1:1000 Total Akt Cell Signaling Cat. #9272 1:1000 pFOXO1 Ser 256 Sigma Aldrich Cat. #SAB4300094-100UG 1:1000 Total FOXO1 Cell Signaling Cat. #2880 1:1000 pIRS1 Ser 307 Thermo Fisher Scientific Cat. #PA1-1054 1:1000 Total IRS1 Upstate Cat. #06-248 1:1000 pIRS2 Ser 731 GeneTex Cat. #GTX23690 1:1000 Total IRS2 Upstate Cat. #06-506 1:1000 pIRb Tyr 1150/1151 Cell Signaling Technology Cat. #3024S 1:1000 Total insulin receptor b Cell Signaling Technology Cat. #3025S 1:1000 pmTOR Ser 2448 Cell Signaling Technology Cat. #2971 1:1000 Total mTOR Cell Signaling Technology Cat. #2972S 1:1000 pP70S6K Thr 389 Cell Signaling Technology Cat. #9205S 1:3000 Total P70S6K Cell Signaling Technology Cat. #9202 1:1000 FGF15 Santa Cruz Biotechnology Cat. #sc-514647 1:1000 pFOXO1 Ser 256 (IHC) Sigma-Aldrich Cat. #SAB4300094-100UG 1:200 IHC, immunohistochemistry.

Techniques: Control, Western Blot, Saline