Journal: Shock (Augusta, Ga.)

Article Title: Restorative mechanisms regulating protein balance in skeletal muscle during recovery from sepsis

doi: 10.1097/SHK.0000000000000762

Figure Lengend Snippet: A-F: Representative Western blots and quantitation are presented for phosphorylated and/or total Akt, PRAS40, TSC2, AMPK, and REDD1. The final panel is a representative tubulin blot demonstrating equal protein loading. Loading controls (tubulin or actin) were run for all proteins examined, but only representative data are shown. Bar graphs: were quantified for phosphorylation of each protein normalized to the total amount of the respective protein, except for REDD1 where total protein was normalized to tubulin. Control values were set to 100 arbitrary units (AU). Values are mean ± SEM. *P < 0.05 sepsis-recovery compared to pair-fed controls values; n = 17 and 10, respectively.

Article Snippet: Antibodies included (Cell Signaling, Beverly, MA, unless otherwise noted): S6K1, S6K1 (Thr389), rpS6, rpS6 (Ser240/244 and Ser230/235), 4E-BP1 (Bethyl Laboratories, Montgomery, TX), 4E-BP1 (Ser65), eukaryotic elongation factor (eEF)2, eEF2 (Thr56), eEF2 kinase, eEF2K (Ser366; Dr. Chris Proud), ERK (extracellular signal-regulated kinses), ERK1/2 (Thr202/Tyr204), RSK (90 kDa ribosomal S6 kinase), RSK1/2 (Ser380), REDD1 (regulated in development and DNA damage responses; ProteinTech, Chicago, IL), Akt, Akt (Thr308 and Ser473), PRAS40 (proline-rich Akt substrate of 40 kDa), PRAS40 (Thr246), eIF4B, eIF4B (Ser422), AMP-activated protein kinase-α (AMPK), AMPKα (Thr172), tuberous sclerosis complex 2 (TSC2), TSC2 (Thr1462), Unc-51 like autophagy activating kinase 1 (ULK1), ULK1 (Ser757), p62 (aka SQSTM1), light-chain 3B (LC3B)-I and –II, and MyoD and myogenin.

Techniques: Western Blot, Quantitation Assay

Journal: Journal of Cancer

Article Title: Gambogic acid affects ESCC progression through regulation of PI3K/AKT/mTOR signal pathway

doi: 10.7150/jca.41115

Figure Lengend Snippet: Expression of apoptosis-related proteins and PI3K/AKT/mTOR pathway components in ESCC cells incubated with GA. (A) The expression levels of cleaved-PARP1, Bcl-2, Bax, cleaved-caspase 3 and cleaved-caspase9 were detected by using western blot analysis. (B) Western blotting was performed to analyze the levels of PI3K, p-AKT, p-mTOR, AKT, mTOR and PTEN.

Article Snippet: The following primary antibodies were applied in the experiments: cleaved-caspase3, cleaved-caspase9, cleaved-PARP1, phosphorylated AKT, Bcl-2, MMP2, MMP9 and β-actin (Proteintech Group, Wuhan, China); PARP1, BAX, PI3K, total AKT, phosphorylated AKT (ser473), p-mTOR (ser2448), cyclinB1 and cyclinD1 (Cell Signaling Technology, MA, USA). β-actin was chosen as a loading control in all experiments.

Techniques: Expressing, Incubation, Western Blot

Journal: Oncotarget

Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

doi: 10.18632/oncotarget.12207

Figure Lengend Snippet: A. IGF-1 concentrations were determined by ELISA in SW620 and SW620 EGFR cell culture media. SW620 cells were cultured to 50% confluence and then transfected with human pCDNA6 vector or pCDNA6-EGFR WT plasmid. After 24 h, cells were transferred to normal medium and cultured for an additional 24 h. Conditioned medium from SW620 cells was collected, centrifuged at 3000 rpm for 10 minutes, and stored at −80°C until use. B. IGF-1 concentrations in HCT116 and HCT116 EGFR (left) and HCT116 and HCT116 KO-EGFR (right) cell culture media were measured by ELISA. C. IGF-1 mRNA levels in HCT116, HCT116 EGFR, and HCT116 siEGFR cells were measured by q-PCR. D. IGF-1 mRNA levels in normal, 2AD, and 2AD+cetu mouse tissues were measured by q-PCR. E. Arg1 protein levels were detected by Western blot in Ana-1 macrophages after treatment with mouse recombinant IGF1 (0, 50, 100, or 200 ng/mL) for 48 h. F. Levels of IGF1R signaling pathway-related proteins were detected by Western blot. Ana-1 cells were harvested after treatment with 100 ng/mL mouse recombinant IGF-1 for 5 or 10 min, or after pretreatment with AG1024 for 30 min followed by 100 ng/mL mouse recombinant IGF-1 for 5 or 10 min. G. Protein levels were detected by Western blot in Ana-1 cells. Ana-1 cells were pretreated with AG1024 for 30 min, and medium was then replaced with fresh RPMI 1640 with AG1024 (10 mM) or HCT116 CM with AG1024 (10 mM) followed by an additional 48 h of culture. Cells were harvested and levels of Arg1, iNOS, and IGF1R signaling pathway-related proteins p-IRS(Tyr632), IGF1R, p-p44/42 MAPK(T202/Y204), p-44/42 MAPK(Erk1/2), p-Akt(Thr308), and Akt were measured by Western blot. Bars represent means ± SD (n = 3) for each treatment. * p < 0.05; ** p < 0.01.

Article Snippet: The antibodies included β-actin (Sigma-Aldrich), p-IGF1R (Tyr1165/1166), Arginase1 (H-52), NOS2 (N-20), and p-IRS (Tyr632) (Santa Crus Biotech., Santa Cruz, CA, USA), and p-EGFR (Tyr1068), p-p44/42 MAPK (T204/Y202), EGFR, Akt, p44/42 MAPK (Erk1/2), IGF1R (CST), and p-Akt (Thr308) (Affinity Biosciences Inc.)

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, Plasmid Preparation, Western Blot, Recombinant

Journal: Frontiers in Cardiovascular Medicine

Article Title: Irisin Suppresses Nicotine-Mediated Atherosclerosis by Attenuating Endothelial Cell Migration, Proliferation, Cell Cycle Arrest, and Cell Senescence

doi: 10.3389/fcvm.2022.851603

Figure Lengend Snippet: Irisin inhibits nicotine-mediated endothelial cell migration and proliferation. (A) Wound healing assay under nicotine, irisin and nicotine + irisin interventions performed at 0, 6 and 12 h during phase II intervention, n = 3. (B) Migration rate among groups at 6 and 12 h during phase II administration and 2 phases treatment pattern illustration. (C) CCK-8 cell proliferation test performed at 6–24 h during phase II intervention, n = 4. (D) Immunoblotting bands of PI3K, AKT, p-AKT (S473), GAPDH, and β-actin. (E) Quantification of PI3K, AKT, and p-AKT. All results were normalized to the expression level of GAPDH or β-actin and presented as the fold change of the Control group, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data are mean ± SD; ns, no significance.

Article Snippet: The primary antibodies used for immunoblotting were PI3K (1:4,000, YM3408, Immunoway, China), AKT (1:4,000, 4691, CST, United States), p-AKT (S473, 1:4,000, YP0006, Immunoway, China), P53 (1:4,000, Ab26, Abcam, China), P21 (1:2,000, ab109199, Abcam, China), P16 INK 4 a (1:5,000, ab108349, Abcam, China), GAPDH (1:5,000, YM3029, Immunoway, China), and β-Actin (1:5,000, YM3028, Immunoway, China).

Techniques: Migration, Wound Healing Assay, CCK-8 Assay, Western Blot, Expressing