primary antibodies specific to p-erk1/2, ekr1/2, p-p38 mapk, p38 mapk, p-akt, akt, cyclin d1, and β-actin  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: Photodynamically tunable ROS-generating hydrogels for accelerated tissue regeneration

doi: 10.1016/j.bioactmat.2025.05.006

Figure Lengend Snippet: Assessment of fibroblast response to ROS-induced effects (A) Cellular proliferation assay using NHDF cells subjected to LED irradiation (100 μW/cm 2 ) for different durations. (i) Non-irradiation, (ii) 20 min, (iii) 30 min, (iv) 40 min, (v) 50 min, (vi) 60 min LED irradiation. ∗p < 0.05 vs. HA (n = 5). (B) Estimated intracellular ROS levels at different irradiation time points under 100 μW/cm 2 intensity. #p < 0.01 vs. con (n = 3). (C) Cells were harvested after 24, 48, and 72 h after LED irradiation. p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, p-AKT, AKT, cyclin D1 and β-actin proteins levels were measured using Western blot. The graphs show quantified proteins levels; (i) relative p-ERK1/2 level, (ii) p-ERK1/2/ERK1/2, (iii) relative p-p38 MAPK level, (iv) p-p38 MAPK/p38 MAPK, (v) relative p-AKT level, (vi) p-AKT/AKT (vii) relative cyclin D1 level. Data are expressed as means ± SD (n = 3). ∗p < 0.05 vs. 24 h HA (D) (i) Total collagen estimated using the Sircol soluble collagen assay kit. (ii) Collagen amount normalized to total protein content showing increased collagen level due to the higher fibroblast number. ∗p < 0.05 vs. HA (n = 3). Throughout all figures and graphs in the in vitro experiments, HA represents the group using HA hydrogel, while CH denotes the group using Ce6-HA hydrogel. This labeling is consistently applied across all in the in vitro data representations in the manuscript.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C with gentle agitation in a 5 % BSA solution containing primary antibodies specific to p-ERK1/2, EKR1/2, p-p38 MAPK, p38 MAPK, p-AKT, AKT, cyclin D1, and β-actin (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Proliferation Assay, Irradiation, Western Blot, Collagen Assay, In Vitro, Labeling

Journal: eLife

Article Title: Stabilization of GTSE1 by cyclin D1–CDK4/6-mediated phosphorylation promotes cell proliferation with implications for cancer prognosis

doi: 10.7554/eLife.101075

Figure Lengend Snippet: ( A ) Schematic of the bioinformatics workflow implemented to identify potential substrates of the D-type cyclins.The process integrates proteomic data from AMBRA1 knockout (KO) U2OS cells versus parental cells , protein annotations of the CDK phosphorylation consensus site [S/T*]PX[K/R] from PhosphoSitePlus , and cancer-related databases. ( B ) Kaplan–Meier curves representing the overall survival analysis based on the 50% upper versus lower expression levels of GTSE1. Survival analysis was conducted across the indicated cancer cohorts. The hazard ratio (HR) was calculated to estimate the relative risk, and significance was assessed using the log-rank test with a threshold of p < 0.05. Curves were generated using the GEPIA2 platform .

Article Snippet: The following antibodies were used: GTSE1 (1:1000, Bethyl Laboratories #A302-274A), β-actin (1:5000, Sigma-Aldrich A5441), AMBRA1 (1:1000, Proteintech Group 13762-1-AP), cyclin A (1:5000, M.P. laboratory), cyclin B1 (1:5000, M.P. laboratory), cyclin D1 (1:1000, Abcam ab16663), p-cyclin D1 (T286) (1:1000, Cell Signaling Technology 3300S), cyclin E (1:1000, Santa Cruz Biotechnology sc-247), Flag (1:2000, Sigma-Aldrich F1804), Flag (1:2000, Sigma-Aldrich F7425), GST (1:5000, GE Healthcare 27457701), HA (1:2000, Bethyl Laboratories A190-108A), LC-3 (1:5000, Novus Biological NB100-2220), p21 (1:1,000, Cell Signaling Technology 2947S), p27 (1:1000, BD Biosciences 610241), p62 (1:5000, MBL International PM045), RB (1:1000, Cell Signaling Technology 9313S), RB (1:1000, Cell Signaling Technology 9309S), p-RB (S807/811) (1:1000, Cell Signaling Technology 9308S), SKP1 (1:5,000, M.P. laboratory), and α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Knock-Out, Phospho-proteomics, Expressing, Generated

Journal: eLife

Article Title: Stabilization of GTSE1 by cyclin D1–CDK4/6-mediated phosphorylation promotes cell proliferation with implications for cancer prognosis

doi: 10.7554/eLife.101075

Figure Lengend Snippet: ( A ) Immunoblot analysis following transient transfection of the indicated proteins in HEK293T cells detailing their phosphorylation status via differential mobility in phos-tag gels in the presence or absence of cyclin D1–CDK4 co-expression. ( B, C ) The indicated purified, recombinant proteins were incubated in the presence or absence of recombinant cyclin D1–CDK4 complex. ERK2 was used as a negative control. Post-incubation, differential phosphorylation was analyzed using phos-tag gels to detect mobility shifts. Immunoblot analysis was conducted to verify the presence of the recombinant proteins as indicated. ( D ) HEK293T cells transfected with either the indicated Flag-tagged proteins or an empty vector (EV). Cell lysates were subjected to immunoprecipitation followed by immunoblot analysis. Inputs (5%) represent whole-cell extracts before pull down. ( E ) Schematic representation of candidate CDK phosphorylation sites in GTSE1, as indicated by the PhosphoSitePlus database . ( F ) HEK293T cells were transiently transfected with the indicated HA-GTSE1 mutants, in the presence or absence of cyclin D1–CDK4 co-expression. Changes in protein migration were analyzed by phos-tag gels. ( G ) Immunoblot and phos-tag gel analysis displaying cell cycle synchronization effects following 72 hr of serum starvation in parental T98G cells and AMBRA1 KO T98G cells. Cells were collected at various time points post-serum re-supplementation as indicated, followed by immunoblotting with the indicated antibodies. ( H ) HA-tagged wild-type GTSE1 (left) or GTSE1 mutated at positions 91, 261, 454, and 724 (Tetra SA) (right) was subjected to co-expression with various cyclins and CDKs, in the presence or absence of the indicated kinase inhibitors to observe the impact on phosphorylation. ( I ) Box plot showing GTSE1 phosphorylation levels normalized to total GTSE1 protein abundance across a pan-cancer cohort relative to adjacent normal tissue, utilizing data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) . Statistical significance was assessed using the Wilcoxon rank-sum test, with a p-value threshold of <0.05. ( J ) Heatmap illustrating differential abundance in cancer of the three GTSE1 phosphorylation sites found in the current study using CPTAC data . Color intensity reflects the log2-transformed Z -scores of identified GTSE1 phospho-peptides, indicating relative phosphorylation levels across various tumor cohorts compared to adjacent normal tissues. The complete analysis of all phosphorylation sites can be found in . Figure 2—source data 1. Original files for western blot analysis displayed in . Figure 2—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 2—source data 3. The raw data for the box plot in depicting GTSE1 phosphorylation levels normalized to total GTSE1 protein abundance across a pan-cancer cohort is provided as log2(Intensities) across samples for global proteins and phospho-proteins of interest. Figure 2—source data 4. The pre-processed data for the box plot in and the heatmap illustrating differential abundance of GTSE1 phosphorylation sites in is provided as Row Z -score of GTSE1 log2(Intensities) across samples for global protein and phospho-protein.

Article Snippet: The following antibodies were used: GTSE1 (1:1000, Bethyl Laboratories #A302-274A), β-actin (1:5000, Sigma-Aldrich A5441), AMBRA1 (1:1000, Proteintech Group 13762-1-AP), cyclin A (1:5000, M.P. laboratory), cyclin B1 (1:5000, M.P. laboratory), cyclin D1 (1:1000, Abcam ab16663), p-cyclin D1 (T286) (1:1000, Cell Signaling Technology 3300S), cyclin E (1:1000, Santa Cruz Biotechnology sc-247), Flag (1:2000, Sigma-Aldrich F1804), Flag (1:2000, Sigma-Aldrich F7425), GST (1:5000, GE Healthcare 27457701), HA (1:2000, Bethyl Laboratories A190-108A), LC-3 (1:5000, Novus Biological NB100-2220), p21 (1:1,000, Cell Signaling Technology 2947S), p27 (1:1000, BD Biosciences 610241), p62 (1:5000, MBL International PM045), RB (1:1000, Cell Signaling Technology 9313S), RB (1:1000, Cell Signaling Technology 9309S), p-RB (S807/811) (1:1000, Cell Signaling Technology 9308S), SKP1 (1:5,000, M.P. laboratory), and α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Western Blot, Transfection, Phospho-proteomics, Expressing, Purification, Recombinant, Incubation, Negative Control, Plasmid Preparation, Immunoprecipitation, Migration, Quantitative Proteomics, Transformation Assay

Journal: eLife

Article Title: Stabilization of GTSE1 by cyclin D1–CDK4/6-mediated phosphorylation promotes cell proliferation with implications for cancer prognosis

doi: 10.7554/eLife.101075

Figure Lengend Snippet: ( A ) HEK293T cells transfected with the indicated single Ser-to-Ala mutants of HA-tagged GTSE1, with and without cyclin D1–CDK4 co-expression, followed by analysis of their migration pattern on phos-tag gels. ( B ) Immunoblot analysis displaying cell cycle synchronization effects following 72 hr of serum starvation in parental T98G cells and AMBRA1 KO T98G cells. Cells were collected at various time points post-serum re-supplementation as indicated, followed by immunoblotting with the indicated antibodies. Cyclin A2 and cyclin B1 serve as S phase and G2-M markers, respectively. pH 3 at Ser10 serves as a mitotic marker. GAPDH serves a loading control. ( C ) HEK293T cells were subjected to transfection with the indicated variants of HA-tagged GTSE1, with and without FFSS–Cyclin D1–CDK4 co-expression. Where indicated, cells were treated with Palbociclib for 4 hr before harvest. Finally, samples were subjected to western blot analysis with the indicated antibodies. ( D ) Heatmap illustrating differential abundance of GTSE1 phosphorylation in cancer using Clinical Proteomic Tumor Analysis Consortium (CPTAC) data . Color intensity reflects the log2-transformed Z -scores of identified GTSE1 phospho-peptides, indicating relative phosphorylation levels across various tumor cohorts compared to adjacent normal tissues. ( E ) HCT mAID FLAG-AMBRA KI cells underwent pull-down using an anti-FLAG antibody versus an IgG sham pull-down. Where indicated, cells were treated with MLN4924 for 3 hr prior to harvesting. Western blot analysis was performed using the specified antibodies to compare the pull-down fraction with the whole-cell extract (WCE). Vinculin serves as WCE loading control. Figure 3—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 3—figure supplement 1—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 3—figure supplement 1—source data 3. The complete analysis for of all GTSE1 phosphorylation sites is provided as Row Z -score of GTSE1 log2(Intensities) across samples for global protein and phospho-protein.

Article Snippet: The following antibodies were used: GTSE1 (1:1000, Bethyl Laboratories #A302-274A), β-actin (1:5000, Sigma-Aldrich A5441), AMBRA1 (1:1000, Proteintech Group 13762-1-AP), cyclin A (1:5000, M.P. laboratory), cyclin B1 (1:5000, M.P. laboratory), cyclin D1 (1:1000, Abcam ab16663), p-cyclin D1 (T286) (1:1000, Cell Signaling Technology 3300S), cyclin E (1:1000, Santa Cruz Biotechnology sc-247), Flag (1:2000, Sigma-Aldrich F1804), Flag (1:2000, Sigma-Aldrich F7425), GST (1:5000, GE Healthcare 27457701), HA (1:2000, Bethyl Laboratories A190-108A), LC-3 (1:5000, Novus Biological NB100-2220), p21 (1:1,000, Cell Signaling Technology 2947S), p27 (1:1000, BD Biosciences 610241), p62 (1:5000, MBL International PM045), RB (1:1000, Cell Signaling Technology 9313S), RB (1:1000, Cell Signaling Technology 9309S), p-RB (S807/811) (1:1000, Cell Signaling Technology 9308S), SKP1 (1:5,000, M.P. laboratory), and α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Transfection, Expressing, Migration, Western Blot, Marker, Control, Phospho-proteomics, Transformation Assay

Journal: eLife

Article Title: Stabilization of GTSE1 by cyclin D1–CDK4/6-mediated phosphorylation promotes cell proliferation with implications for cancer prognosis

doi: 10.7554/eLife.101075

Figure Lengend Snippet: ( A ) Immunoblot analysis of whole-cell extracts from parental HCT-116 cells, AMBRA1 knockout (KO) HCT-116 cells, and HCT-116 cells expressing either wild-type cyclin D1 or an AMBRA1-insensitive mutant of cyclin D1 (T286A), in the presence or absence of the CDK4/6 inhibitor Palbociclib. ( B ) Immunoblot analysis of whole-cell extracts from HCT-116 cells harboring an endogenous mini-AID domain fused to AMBRA1 N-terminus. Cells were subjected to either incubation with auxin and doxycycline to induce AMBRA1 degradation or transfection with cyclin D1(T286A) in the presence or absence of Palbociclib. ( C ) Cycloheximide (CHX) chase assay in HCT-116 cells with mAID-AMBRA1, treated with or without auxin and doxycycline to induce AMBRA1 degradation. Immunoblot analyses were conducted to assess the stability of GTSE1 and other indicated proteins, with the stable protein SKP1 used as a loading control. ( D ) Protein stability assessment via CHX chase in U2OS parental cells and AMBRA1 KO clones (C11 and G11). The protein levels of AMBRA1, GTSE1, and cyclin D1 were analyzed by immunoblotting, with tubulin serving as a loading control. ( E ) U2OS cells stably transduced with retroviruses expressing the indicated GTSE1 constructs were subjected to CHX chase assays. Subsequent immunoblotting was conducted for the indicated proteins, with tubulin utilized as a loading control. ( F ) Densitometric quantification of GTSE1 band intensity from ( E ) and two identical experiments normalized using tubulin. Initial band intensity at time 0 is set as the 100% reference point. Error bars represent SEM ( n = 3 of biological replicates). ( G ) Time-lapse microscopy images of U2OS cells stably expressing EGFP-tagged the indicated GTSE1 constructs during a CHX chase. The EGFP signal intensity corresponds to GTSE1 levels, while cells are stained with a far-red cell tracker for cell masking. Scale bars indicate 120 μM. ( H ) Quantitative analysis of EGFP fluorescence intensity from time-lapse experiment shown in ( G ) plus two identical experiments. The initial fluorescence intensity was normalized to 100% at time zero. Data represent the mean fluorescence intensity from the three independent measurements, with error bars indicating SEM.( I ) U2OS cells were treated with various inhibitors for 3 hr before harvest: the proteasome inhibitor MG132, the CRL inhibitor MLN4924, and the V-ATPase inhibitor Bafilomycin A1. Immunoblot analysis of the indicated proteins was performed, with tubulin as a loading control. p62 and LC3 serve as autophagy inhibition controls, while p27 is used as control for CRL- and ubiquitin–proteasome system (UPS)-dependent degradation. Figure 3—source data 1. Original files for western blot analysis displayed in . Figure 3—source data 2. PDF file containing original western blots for , indicating the relevant bands.

Article Snippet: The following antibodies were used: GTSE1 (1:1000, Bethyl Laboratories #A302-274A), β-actin (1:5000, Sigma-Aldrich A5441), AMBRA1 (1:1000, Proteintech Group 13762-1-AP), cyclin A (1:5000, M.P. laboratory), cyclin B1 (1:5000, M.P. laboratory), cyclin D1 (1:1000, Abcam ab16663), p-cyclin D1 (T286) (1:1000, Cell Signaling Technology 3300S), cyclin E (1:1000, Santa Cruz Biotechnology sc-247), Flag (1:2000, Sigma-Aldrich F1804), Flag (1:2000, Sigma-Aldrich F7425), GST (1:5000, GE Healthcare 27457701), HA (1:2000, Bethyl Laboratories A190-108A), LC-3 (1:5000, Novus Biological NB100-2220), p21 (1:1,000, Cell Signaling Technology 2947S), p27 (1:1000, BD Biosciences 610241), p62 (1:5000, MBL International PM045), RB (1:1000, Cell Signaling Technology 9313S), RB (1:1000, Cell Signaling Technology 9309S), p-RB (S807/811) (1:1000, Cell Signaling Technology 9308S), SKP1 (1:5,000, M.P. laboratory), and α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Western Blot, Knock-Out, Expressing, Mutagenesis, Incubation, Transfection, Control, Clone Assay, Stable Transfection, Transduction, Construct, Time-lapse Microscopy, Staining, Fluorescence, Inhibition, Ubiquitin Proteomics