Journal: PLOS ONE

Article Title: Investigation of the anti-tumor mechanism of tirabrutinib, a highly selective Bruton’s tyrosine kinase inhibitor, by phosphoproteomics and transcriptomics

doi: 10.1371/journal.pone.0282166

Figure Lengend Snippet: Significantly downregulated phosphosites in TMD8 cells.

Article Snippet: Rabbit monoclonal anti- phosphorylated BTK (Tyr-223) antibody (p-BTK; Novus Biologicals, #NB100-79907), rabbit monoclonal BTK antibody (Cell Signaling Technology, Inc., #3533), rabbit polyclonal phosphorylated AKT (Ser-473) antibody (p-AKT; Cell Signaling Technology, Inc., #9271), rabbit monoclonal AKT (pan) (C67E7) antibody (Cell Signaling Technology, Inc., #4691), rabbit polyclonal phosphorylated p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204) antibody (p-ERK1/2; Cell Signaling Technology, Inc., #9101), rabbit monoclonal p44/42 MAPK (ERK1/2) (137F5) antibody (Cell Signaling Technology, Inc., #4695), rabbit monoclonal phosphorylated PLCγ2 (Tyr-759) (E9E9Y) antibody (p-PLCγ2; Cell Signaling Technology, Inc., #50535), rabbit monoclonal PLCγ2 (E5U4T) antibody (Cell Signaling Technology, Inc., #55512), mouse monoclonal phosphorylated IκBα (Ser-32/36) (5A5) antibody (p-IκBα; Cell Signaling Technology, Inc., #9246), rabbit monoclonal IκBα (44D4) antibody (Cell Signaling Technology, Inc., #4812), rabbit polyclonal phosphorylated PKCβ (phospho-Thr-641) antibody (p-PKCβ; Signalway Antibody LLC, #11172), rabbit monoclonal IRF4 (D9P5H) antibody (Cell Signaling Technology, Inc., #15106), rabbit monoclonal BCL6 (D65C10) antibody (Cell Signaling Technology, Inc., #5650), rabbit monoclonal c-MYC (D84C12) antibody (Cell Signaling Technology, Inc., #5605), and rabbit monoclonal GAPDH (14C10) antibody (Cell Signaling Technology, Inc., #2118) were used.

Techniques: Sequencing, Translocation Assay, Variant Assay

Journal: The Journal of Experimental Medicine

Article Title: SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

doi: 10.1084/jem.20220906

Figure Lengend Snippet: Spike suppresses signaling at an early step of IS assembly. (A–C) Left: Quantification (%) of 5-min and 15-min SAg-specific conjugates harboring PTyr (A), CD3ζ (B), or P-ZAP-70 (C) staining at the IS in CTLs (day 7) pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W, then mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 5 or 15 min at 37°C (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Relative PTyr (A), CD3ζ (B), or P-ZAP-70 (C) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). (D) Flow cytometric analysis of protein tyrosine phosphorylation in conjugates prepared as in A–C and incubated at 37°C for the indicated times. Raji cells were loaded with 1.5 μM CFSE prior to conjugate formation. Conjugates were stained with anti-PTyr mAb followed by fluorescently labeled secondary Abs. The analysis was carried out gating on CSFE − cells ( n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05). (E) Immunoblot analysis of Erk phosphorylation in CTLs activated with anti-CD3 and anti-CD28 mAbs. CTLs were incubated at 37°C for the indicated times and processed for immunoblot with Abs against the active forms of Erk1/2. Stripped filters were blotted with anti-Erk2 mAb as loading control. The migration of molecular mass markers is indicated ( n = 2, one-way ANOVA test; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Data are expressed as mean ± SD. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Nonsignificant differences are not shown. Source data are available for this figure: .

Article Snippet: Immunoblotting was carried out using anti-phospho-Erk1/2 ([1:500], phosphorylated p44/42 [MAPK] Erk1/2, #9101; Cell Signaling), anti-Erk2 ([1:500], clone D2, #sc-1647; SantaCruz), anti-ACE2 ([1:500], clone SN0754, #MA5-32307; Invitrogen), and anti-actin ([1:10,000], #MAB1501; Merck Millipore) Abs and peroxidase-labeled secondary Ig, and a chemiluminescence detection kit (Pierce Rockford).

Techniques: Staining, Incubation, Fluorescence, Labeling, Western Blot, Migration

Journal: Pharmaceuticals

Article Title: Renin–Angiotensin System Antagonism Protects the Diabetic Heart from Ischemia/Reperfusion Injury in Variable Hyperglycemia Duration Settings by a Glucose Transporter Type 4-Mediated Pathway

doi: 10.3390/ph16020238

Figure Lengend Snippet: Immunoblotting showing the effects of Captopril, Losartan, or their combination on ERK1/2 and eNOS phosphorylation levels in the left ventricle homogenate ( n = 4 per group). Western blot evaluations showing ERK1/2 and Phosphorylated ERK1/2 (P-ERK1/2) ( A – C ), eNOS and Phosphorylated eNOS (P-eNOS) ( D – F ). ( A , D ), hyperglycemia; ( B , E ), four weeks diabetic hearts; ( C , F ), six weeks diabetic hearts. Values are presented as means ± SEM. Ctr, control; Cap, Captopril; Los, Losartan; ERK, extracellular signal-regulated protein kinase; and eNOS, nitric oxide synthase.

Article Snippet: The frozen protein samples were used in Western blot analysis to evaluate the protein expression using: anti-superoxide dismutase [Cu-Zn] (SOD) antibody (cat# 07-403-I EMD Millipore, Sigma, St. Louis, Missouri, USA), anti-catalase (CAT) antibody (Cat# 12980); anti-extracellular regulated kinases 1/2 (ERK1/2) antibody (cat# 9102); anti-phosphorylated ERK1/2 antibody (cat #9743); anti-caspase-3 antibody (cat #9662); anti-caspase-8 antibody (cat #4927); anti-phosphorylated endothelial nitric oxide synthase (eNOS) (Ser1177) antibody (cat #9571); anti-eNOS (49G3) antibody (cat #Ab #9586) (all from Cell Signaling Technology, Danvers, Massachusetts, USA) and anti-glucose transporter 1 (GLUT-1) antibody (cat #ab115730, Abcam, Cambridge, UK), anti-glucose transporter 4 (GLUT-4) antibody (phosphor S488) (cat #ab188317 Abcam) by standard immunoblot procedures as previously described [ ].

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Inhibition of autophagy and MEK promotes ferroptosis in Lkb1-deficient Kras-driven lung tumors

doi: 10.1038/s41419-023-05592-8

Figure Lengend Snippet: A Clonogenic survival assay of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with HCQ or Trametinib individually at indicated concentrations. B Cell growth inhibition curve of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with HCQ or Trametinib individually at indicated concentrations in Table . C Clonogenic survival assay of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with the combination of HCQ and Trametinib at indicated concentrations. D Relative proliferation of KL (clone 2126 3-2 and clone 2126 5-5) and KP (clone 2871-7 and clone 2871-8) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. E Western blot for LC3, pERK, total ERK, pS6, total S6 and β-actin of KL and KP TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h. F Scheme of the KL or KP TDCLs for measuring oxygen consumption rate (OCR) using Seahorse XFe24 analyzer. G Basal respiration and ATP production of KL TDCLs (clone 2126 3-2 and clone 2126 5-5 (with black squares)) after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. H Basal respiration and ATP production of KP TDCLs (clone 2871-1 and clone 2871-8 (with black squares)) after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. I Scheme of the metabolomics analysis via LC-MS of KL TDCLs after 6 h’ treatment. J The levels of metabolites of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs after 6 h’ treatment with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination. K Left: Overlapping images of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with MitoTracker Red CMXRos for mitochondrial membrane potential and MitoTracker Green FM for mitochondrial mass. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KL TDCLs quantified by the ratio of red fluorescence intensity and green fluorescence intensity. L Left: Overlapping images of KP TDCLs (clone 2871-7 and clone 2871-8) treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with MitoTracker Red CMXRos for mitochondrial membrane potential and MitoTracker Green FM for mitochondria mass. Blue: Hoechst 33342 for nuclear staining. Right: graph of relative mitochondrial membrane potential of KP TDCLs quantified by the ratio of red fluorescence intensity and green fluorescence intensity. M Left: Overlapping images of KL (clone 2126 3-2 and clone 2126 5-5) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with TMRM (red fluorescence) for mitochondrial membrane potential. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KL TDCLs quantified by the ratio of red fluorescence intensity and total cell numbers. N Left: Overlapping images of KP (clone 2871-1 and clone 2871-8) TDCLs treated with vehicle control, HCQ (10 μM), Trametinib (2.5 nM) and the combination for 6 h and stained with TMRM (red fluorescence) for mitochondrial membrane potential. Blue: Hoechst 33342 for nuclear staining. Right: graph of the relative mitochondrial membrane potential of KP TDCLs quantified by the ratio of red fluorescence intensity and total cell numbers. Data are mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used for Western blots: LC3 (Novus Biologicals, NB600-1384), total ERK (Cell Signaling, 4695), phosphorylated ERK (Cell Signaling, 9101), total S6 (Cell Signaling, 2217), phosphorylated S6 (Cell Signaling, 4858), and β-actin (Sigma, A1978).

Techniques: Clonogenic Cell Survival Assay, Inhibition, Western Blot, Liquid Chromatography with Mass Spectroscopy, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Normoxia- and Hypoxia-Modified Extracellular Vesicle Therapy in Function, Perfusion, and Collateralization in Chronically Ischemic Myocardium

doi: 10.3390/ijms24032076

Figure Lengend Snippet: Hypoxia-modified EV treatment increased pro-angiogenic signaling and decreased anti-angiogenic signaling in chronically ischemic myocardium compared to normoxia EV treatment. Immunoblot results from total protein lysates of chronically ischemic myocardium in swine treated with hypoxia-modified EVs (HYP, n = 7) compared to normoxia serum-starved EVs (NOR, n = 10) are shown for pro-angiogenic markers Akt, phosphorylated (p-) Akt, and the ratio of p-Akt to Akt; extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and the ratio of p-ERK 1/2 to ERK1/2; endothelial nitric oxide synthase (eNOS), p-eNOS, and the ratio of p-eNOS and eNOS; and vascular endothelial cadherin (VE-Cadherin). Immunoblot results are also shown for anti-angiogenic markers angiostatin and endostatin. Upper and lower borders of box represent upper and lower quartiles; middle horizontal line represents median; upper and lower whiskers represent maximum and minimum values. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Total protein (40 µg) was fractionated onto a 4–12% Bis-Tris gel (ThermoFisher Scientific, Waltham, MA, USA), and membranes were incubated overnight at 4 °C with 1:1000 dilutions of individual rabbit polyclonal primary antibodies to Akt (CS9272), phosphorylated (Ser473) Akt (p-Akt) (CS4060), extracellular signal-regulated kinase 1/2 (ERK1/2) (CS4695), phosphorylated ERK1/2 (p-ERK1/2) (CS4370), endothelial nitric oxide synthase (eNOS) (CS32027), phosphorylated (Ser1177) eNOS (p-eNOS) (CS9571), vascular endothelial cadherin (VE-Cadherin) (CS2500) (Cell Signaling, Danvers, MA, USA), endostatin (ab207162) (Abcam, Cambridge, UK), and 1:500 dilution of primary antibody to angiostatin (ab2904) (Abcam, Cambridge, UK) (complete antibody information listed in ).

Techniques: Modification, Western Blot

Journal: Aging (Albany NY)

Article Title: miR-6742-5p regulates the invasion and migration of lung adenocarcinoma cells via mediating FGF8/ERK12/MMP9/MMP2 signaling pathway

doi: 10.18632/aging.204277

Figure Lengend Snippet: miR-6742-5p was involved in regulating the invasion and migration of LUAD via targeting FGF8/ERK1/2/MMP9/MMP2. ( A ) The expression of FGF8, ERK1/2, MMP9 and MMP2 was evaluated by western blot in cells treated with and without RO 67-7476 cells. ( B ) The expression of FGF8, ERK1/2, MMP9 and MMP2 quantification in cells treated with and without RO 67-7476 cells. ** P < 0.01. Relative protein levels of FGF8 (-)**P=0.0009miR-6742-5p OE group vs NC group;**P=0.0028miR-6742-5p KD group vs NC group; Ro 67-7476 **P=0.004miR-6742-5p OE group vs NC group;**P=0.0065miR-6742-5p KD group vs NC group; Relative protein levels of ERK1/2 *P=0.0134miR-6742-5p OE group vs NC group;**P=0.0063miR-6742-5p KD group vs NC group; Relative protein levels of MMP2 **P=0.0066miR-6742-5p OE group vs NC group;**P=0.0017miR-6742-5p KD group vs NC group; Relative protein levels of MMP9 **P=0.0058miR-6742-5p OE group vs NC group;**P=0.0051miR-6742-5p KD group vs NC group.

Article Snippet: After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against FGF8, ERK1/2, phosphorylated (p)-ERK1/2 and MMP9 (Cell Signaling Technology, Danvers, MA, USA) at 4° C overnight, followed by incubation with secondary antibodies at 37° C for 1 h. Finally, protein bands were visualized using ECL reagents (Beyotime).

Techniques: Migration, Expressing, Western Blot

Journal: Pharmaceutics

Article Title: Hyaluronic Acid-Modified Cisplatin-Encapsulated Poly(Lactic-co-Glycolic Acid) Magnetic Nanoparticles for Dual-Targeted NIR-Responsive Chemo-Photothermal Combination Cancer Therapy

doi: 10.3390/pharmaceutics15010290

Figure Lengend Snippet: The hematoxylin and eosin (H&E) staining and the immunohistochemical (IHC) staining of phospho-ERK and cleaved caspase-3 in the tumor apoptotic area after different treatments (bar = 100 μm).

Article Snippet: The standard protocol of Western blot was used with rabbit anti-mouse primary antibodies for β-actin (4970L, Cell Signaling, 1:1000), extracellular-signal-regulated kinase (ERK) (MA5-15134, Invitrogen, 1:1000), phosphorylated extracellular-signal-regulated kinase (phospho-ERK) (4376S, Cell Signaling, 1:1000), caspase-3 (GTX110543, GeneTex, 1:1000), and cleaved caspase-3 (9661S, Cell Signaling, 1:1000).

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry