mouse cdk6 monoclonal antibody  (Proteintech)

Journal: eLife

Article Title: Kinases in motion: impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/elife.94755

Figure Lengend Snippet: Figure 4. CDK4/6 interactions and conformations. (A) Illustration of regulatory CDK4/6 interactions and Rb activation. (B) Domain organization of CDK4, CDK6, and p16INK4a; tested point mutations are listed. (C) 3D structure of CDK6 in complex with p16INK4a. Crucial amino acids involved in the interaction of the two proteins are highlighted. The R31C mutant is depicted in orange (PDB code: 1BI7, Russo et al., 1998). (D) Protein:protein interaction (PPI) reporter analyses of the kinases CDK4 and CDK6 with p16INK4a. Scheme illustrates CDK4/6 hetero-dimer formation with p16INK4a analyzed using a PCA RLuc PPI reporter system. PPI induces the complementation of RLuc PCA fragments promoting an increase in bioluminescence (HEK293T cells, 48 hr of transient reporter expression). Bars represent the RLU fold change of PPI in relation to wild-type CDK4/6:p16INK4a complex (mean ± SEM, n=7 ind. experiments). (E) Basal signal of CDK4/6 KinCon reporters with indicated mutations are shown (expressed for 48 hr in HEK293T cells) (mean

Article Snippet: DOI: https://doi.org/10.7554/eLife.94755 20 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct mouse pcDNA3.1CDK6- F[1] CDK6- PPI reporter Transfected construct mouse pcDNA3.1CDK6- R31C- F[1] CDK6- PPI reporter (R31C) Transfected construct mouse pcDNA3.1CDK4- F[1] CDK4- PPI reporter Transfected construct mouse pcDNA3.1CDK4- R24C- F[1] CDK4- PPI reporter (R24C) Transfected construct mouse pcDNA3.1p16INK4a- F[2] p16INK4a- PPI reporter Transfected construct mouse pcDNA3.1- p16INK4a- P40L- F[2] p16INK4a- PPI reporter (P40L) Transfected construct human pcDNA3.1F[1]-MEK1- F[2] MEK1- KinCon Transfected construct human pcDNA3.1- F[1]-MEK1- K57E- F[2] MEK- KinCon (K57E) Transfected construct human pcDNA3.1F[1]-PKAc- F[2] PKAc- KinCon Transfected construct human pcDNA3.1F[1]-PKAc- L206R- F[2] PKAc- KinCon (L206R) Transfected construct human pcDNA3.1MO25- 3xFlag MO25- 3xFlag Transfected construct human pcDNA3.1F[1]-BRAF- F[2] BRAF- KinCon Transfected construct human pcDNA3.1F[1]-BRAF- V600E- F[2] BRAF- KinCon (V600E) Transfected construct human pcDNA3.1F[1]-RIPK1- F[2] RIPK1- KinCon Transfected construct human pcDNA3.1F[1]-RIPK2- F[2] RIPK2- KinCon Transfected construct human pcDNA3.1F[1]-RIPK3- F[2] RIPK3- KinCon Antibody GAPDH Rabbit monoclonal Cell Signaling 2118 1:10000 Antibody AMPKα Rabbit monoclonal Cell Signaling 2532 1:1000 Antibody PhosphoAMPKα(Thr172) Rabbit monoclonal Cell Signaling 2535 1:1000 Antibody LKB1 Rabbit monoclonal Cell Signaling 3047 1:1000 Antibody Vinculin Rabbit monoclonal Cell Signaling 4650 1:1000 Antibody RIP XP Rabbit monoclonal Cell Signaling 3493 1:1000 Antibody RIP3 Rabbit monoclonal Cell Signaling 13526 1:1000 Antibody CDK6 Mouse monoclonal Cell Signaling 3136 1:1000 Continued Continued on next page Kugler, Schwaighofer et al. eLife 2024;13:RP94755.

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: eLife

Article Title: Kinases in motion: impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/elife.94755

Figure Lengend Snippet: Figure 5. Impact of small molecules and protein interactions on kinase activity conformations. (A+B) Depiction of molecular interactions of a type I 1/2 and type III kinase inhibitors with a kinase domain (N and C lobe). Impact of PLX8394, Cobimetinib and GSK547 on wild-type (wt) and mutated versions of BRAF, RIPK1, and MEK1 kinase conformation (KinCon) reporters. 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 1 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4/5 ind. experiments). (C) Depiction of molecular interactions of a type I kinase inhibitor with a kinase domain (N and C lobe). Impact of Abemaciclib on indicated CDK6 kinase conformations (wt and p16INK4a binding deficient). 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 3 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). (D) Bioluminescence measurement of PKAc wt and L206R KinCon reporters. HEK293T cells expressing the reporter were treated with 20 μM of Forskolin for 15 min followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). (E) Kinome tree displays kinases for which KinCon reporters have been generated (red dots). The blue squares highlight the kinases for

Article Snippet: DOI: https://doi.org/10.7554/eLife.94755 20 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct mouse pcDNA3.1CDK6- F[1] CDK6- PPI reporter Transfected construct mouse pcDNA3.1CDK6- R31C- F[1] CDK6- PPI reporter (R31C) Transfected construct mouse pcDNA3.1CDK4- F[1] CDK4- PPI reporter Transfected construct mouse pcDNA3.1CDK4- R24C- F[1] CDK4- PPI reporter (R24C) Transfected construct mouse pcDNA3.1p16INK4a- F[2] p16INK4a- PPI reporter Transfected construct mouse pcDNA3.1- p16INK4a- P40L- F[2] p16INK4a- PPI reporter (P40L) Transfected construct human pcDNA3.1F[1]-MEK1- F[2] MEK1- KinCon Transfected construct human pcDNA3.1- F[1]-MEK1- K57E- F[2] MEK- KinCon (K57E) Transfected construct human pcDNA3.1F[1]-PKAc- F[2] PKAc- KinCon Transfected construct human pcDNA3.1F[1]-PKAc- L206R- F[2] PKAc- KinCon (L206R) Transfected construct human pcDNA3.1MO25- 3xFlag MO25- 3xFlag Transfected construct human pcDNA3.1F[1]-BRAF- F[2] BRAF- KinCon Transfected construct human pcDNA3.1F[1]-BRAF- V600E- F[2] BRAF- KinCon (V600E) Transfected construct human pcDNA3.1F[1]-RIPK1- F[2] RIPK1- KinCon Transfected construct human pcDNA3.1F[1]-RIPK2- F[2] RIPK2- KinCon Transfected construct human pcDNA3.1F[1]-RIPK3- F[2] RIPK3- KinCon Antibody GAPDH Rabbit monoclonal Cell Signaling 2118 1:10000 Antibody AMPKα Rabbit monoclonal Cell Signaling 2532 1:1000 Antibody PhosphoAMPKα(Thr172) Rabbit monoclonal Cell Signaling 2535 1:1000 Antibody LKB1 Rabbit monoclonal Cell Signaling 3047 1:1000 Antibody Vinculin Rabbit monoclonal Cell Signaling 4650 1:1000 Antibody RIP XP Rabbit monoclonal Cell Signaling 3493 1:1000 Antibody RIP3 Rabbit monoclonal Cell Signaling 13526 1:1000 Antibody CDK6 Mouse monoclonal Cell Signaling 3136 1:1000 Continued Continued on next page Kugler, Schwaighofer et al. eLife 2024;13:RP94755.

Techniques: Activity Assay, Transfection, Expressing, Construct, Binding Assay, Generated

Journal: Oncology letters

Article Title: Rapamycin inhibits B16 melanoma cell viability in vitro and in vivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway.

doi: 10.3892/ol.2024.14273

Figure Lengend Snippet: Figure 3. Effects of RAPA on the cell cycle progress of B16‑F10 cells. (A) Cell cycle distribution was assessed by flow cytometry. (B) Results of statistical analysis of cell rates at different time periods for each group in (A). (C) CDK1, cyclin D1, CDK4, CDK6, cyclin E1 and CDK2 protein expression levels were detected by western blotting. Relative expression of (D) CDK1, (E) Cyclin D1, (F) CDK4, (G) CDK6, (H) Cyclin E1 and (I) CDK2 proteins. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. RAPA 0 nM group. ns, not significant; RAPA, rapamycin.

Article Snippet: Mouse GAPDH monoclonal antibody (cat. no. 60004‐1‐Ig), mouse Beclin‐1 monoclonal antibody (cat. no. 66665‐1‐Ig), mouse caspase 3/p17/p19 monoclonal antibody (cat. no. 66470‐2‐Ig), mouse Bax monoclonal antibody (cat. no. 60267‐1‐Ig), mouse Bcl2 monoclonal antibody (cat. no. 68103‐1‐Ig), mouse cyclin D1 monoclonal antibody (cat. no. 60186‐1‐Ig), mouse CDK4 monoclonal anti‐ body (cat. no. 66950‐1‐Ig), mouse CDK6 monoclonal antibody (cat. no. 66278‐1‐Ig), mouse CDK2 monoclonal antibody (cat. no. 60312‐1‐Ig), rabbit cyclin E1 polyclonal antibody (cat. no. 11554‐1‐AP), rabbit microtubule‐associated protein light chain 3 (LC3) polyclonal antibody (cat. no. 14600‐1‐AP), goat anti‐rabbit IgG‐HRP (cat. no. SA00001‐2) and goat anti‐mouse IgG‐HRP (cat. no. SA00001‐1) antibodies were purchased from ProteinTech Group, Inc. Rabbit sequestosome 1/p62 (cat. no. AF5384), mTOR (cat. no. AF6308), p70 ribosomal S6 kinase (p70‐S6k; cat. no. AF6226), eukaryotic initiation factor 4E‐binding protein (4E‐BP1; cat. no. AF6432), phosphorylated (p)‐mTOR (cat. no. AF3308), p‐p70‐S6k (cat. no. AF3228) and p‐4E‐BP1 (cat. no. AF3830) polyclonal antibodies were purchased from Affinity Biosciences.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Animals : an Open Access Journal from MDPI

Article Title: SV40T/E6E7-Induced Proliferation Is Involved in the Activity of E2F3 in Bovine Mammary Epithelial Cells

doi: 10.3390/ani12141790

Figure Lengend Snippet: Involvement of E2F signaling pathway in the proliferation of the immortalized BMECs. ( A ) Western blotting analysis of CDK4, CDK6, cyclin D1, cyclin D3, and CDK2 in primary and immortalized BMECs. ( B ) Western blotting analysis of E2F1, E2F2, and E2F3 in primary and immortalized BMECs. Primary BMECs or BMECs immortalized by E6E7 and SV40T were collected at early (E) and late (L) passages of culture. Data are based on triplicate experiments.

Article Snippet: The following primary antibodies were obtained from Cell Signaling Technology (CST, Shanghai, China): rabbit anti-β-actin monoclonal antibody (diluted 1:1000; #4970), rabbit anti-GAPDH monoclonal antibody (1:1000; #2118), rabbit anti-cyclin D1 monoclonal antibody (1:1000; #2978), rabbit anti-CDK2 monoclonal antibody (1:1000; #2546), mouse anti-CDK6 monoclonal antibody (1:1000; #3136), rabbit anti-cyclin D3 monoclonal antibody (1:1000; #2936), rabbit anti-CDK4 monoclonal antibody (1:1000; #12790), and rabbit anti-p27 Kip1 monoclonal antibody (1:1000; #3686).

Techniques: Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Recreating the Bone Marrow Microenvironment to Model Leukemic Stem Cell Quiescence

doi: 10.3389/fcell.2021.662868

Figure Lengend Snippet: Hypoxia and co-culture with bone marrow mesenchymal stromal cells (BMSC) + TGFβ-1 increases the expression of markers of leukemia cell quiescence. KG1a cells were cultured alone or with alginate-encapsulated BMSC and TGFβ-1 (10 ng/ml) over a 12-day period in normoxia or hypoxia (1% pO 2 ). (A) Expression of CD150 determined in the live fraction by flow cytometry. MFI, mean fluorescent intensity. * p < 0.05 (Student’s t -test). (B–E) Results of quantitative RT-PCRs on RNA isolated on days 8 and 12. * p < 0.05 (one-way ANOVA). (F) Gene expression analysis of p21 expression conducted on the GSE17054 transcriptomic dataset. The graph represents the changes in gene expression between hematopoietic stem cells and leukemic stem cells. * p < 0.05 (Welch’s t -test). (G) Western blot images for the expression of p21, CDK4, and CDK6 conducted on samples isolated at day 12 of culture. SN, single culture at normoxia; CH, co-culture under hypoxia.

Article Snippet: After blocking, the blots were incubated with rabbit monoclonal antibodies against CDK4 [Cell Signaling Technology (CST)] and p21 (Santa Cruz) and with mouse monoclonal antibodies against CDK6 (CST) and actin (Sigma-Aldrich).

Techniques: Co-Culture Assay, Expressing, Cell Culture, Flow Cytometry, Isolation, Gene Expression, Western Blot