protein pull down activation assay kits  (Cytoskeleton Inc)

Journal: Materials Today Bio

Article Title: Nanofibrous topography-driven altered responsiveness to Wnt5a mediates the three-dimensional polarization of odontoblasts

doi: 10.1016/j.mtbio.2022.100479

Figure Lengend Snippet: Nanofiber-induced Wnt5a regulates actin-based filopodia formation through the Cdc42/JNK axis . The activations of the Rho family of small GTPases RhoA (A) and Cdc42 (B) were increased upon the rWNT5a treatment on TCP and FIBER. RhoA and Cdc42 activate each other complimentarily, as determined by a pull-down assay analysis of cells on TCP and FIBER and confirmed by the knockdown of RhoA and Cdc42 (C, D). Immunofluorescence images of MDPC cells showed an increase in the phosphorylation of the JNK level on FIBER and a further increase upon treatment with rWNT5a (E). Schematic illustration of Wnt5a-induced apical polarization of MDPC through the Cdc42/JNK axis (F).

Article Snippet: The activity of small GTPases was determined using a pull-down assay kit following the protocol described by the manufacturer (Cytoskeleton, Denver, CO).

Techniques: Pull Down Assay, Immunofluorescence

Journal: Nature cell biology

Article Title: Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP.

doi: 10.1038/s41556-018-0270-5

Figure Lengend Snippet: Fig. 5 | ECM mechanical cues regulate Lipin-1/ARF1 activity. a,b, qPCR for SREBP target genes in MCF10ATk1 cells treated with DMSO or with 100 μM propranolol (Propra) to inhibit Lipin-1 phosphatidate phosphatase activity (a), or transfected with control (siCo.) and Lipin-1 siRNA (siLipin-1) (b). Data are relative to GAPDH levels; mean expression in controls was set to 1, and all other samples are relative to this (n = 4 biologically independent samples, except DHCR7 and LDLR in b for which n = 6, pooled across two independent experiments for each bar; multiple unpaired two-tailed Student’s t-tests). c, LDLR-luciferase in MDA231 cells transfected with the indicated siRNA and treated with 100 μM propranolol. Mean expression in the control was set to 1, and all other samples are relative to this (n = 4 biologically independent samples pooled across two independent experiments for each bar; unpaired Mann– Whitney tests). d, Immunofluorescence for endogenous SREBP2 in MCF10ATk1 cells treated with propranolol, transfected with Lipin-1 or COPI siRNA, or expressing dominant-negative ARF1-T31N-GFP (DN ARF1). At least 50 cells per condition. e, Co-localization of transfected MYC-SCAP with the GFP-Rab6 Golgi marker in RPE1 cells treated for 6 h with propranolol, in cells transfected with control (siCo.), Lipin-1 or COPI siRNAs, or expressing dominant-negative ARF1-T31N (DN ARF1). At least 50 cells per condition. f, Lipid staining in MCF10ATk1 cells treated for 24 h with YM, with 100 μM propranolol, or transfected with Lipin-1 siRNA. Quantifications and n are provided in Supplementary Table 3. g, Western blotting for Lipin-1 levels in microsomal fractions from MCF10ATk1 cells treated for 3 h with DMSO or Y27632+ML7 (YM). Calreticulin (ER marker, Calret) and GM130 (Golgi marker) are loading controls. h, Co-localization of transfected GFP-PKD-KD with the GM130 Golgi marker in HEK293 cells treated for 30 min with YM, with 100 μM propranolol, or transfected with control (siCo.) and Lipin-1 siRNAs. Mean Pearson’s correlation coefficient and s.d. for co-localization are indicated above each panel (n ≥ 10 cells were measured for each condition). i, GST-GGA3-PBD pulldown for GTP-bound active ARF1 (GTP-Arf1) and western blotting for ARF1 in the total extracts (Total Arf1). Cells were treated 3 h with DMSO or YM. j, Co-localization of endogenous ARF1 with a Golgi marker (GM130) in MCF10ATk1 cells treated 6 h with YM or propranolol. Mean Pearson’s correlation coefficient and s.d. for co-localization are indicated above each panel (n ≥ 10 cells were measured for each condition). Images in d–j are representative of at least two independent experiments with similar results. Scale bars, 10 μm, except for f (5 μm). Data are mean and single points. All n values are pooled across independent experiments. Unprocessed blots are provided in Supplementary Fig. 8.

Article Snippet: Active GTP-bound ARF1 pulldown was performed with a commercial kit following the manifacturer’s intructions (Cytoskeleton BK032).

Techniques: Activity Assay, Transfection, Control, Expressing, Two Tailed Test, Luciferase, MANN-WHITNEY, Immunofluorescence, Dominant Negative Mutation, Marker, Staining, Western Blot

Journal: Cell stem cell

Article Title: Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma

doi: 10.1016/j.stem.2018.02.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GTP-bound RHOA and RAC1 were precipitated using pull-down activation assay Biochem Kits, according to manufacturer’s recommendations (Cytoskeleton).

Techniques: Recombinant, Infection, Western Blot, SYBR Green Assay, Activation Assay, RNA Sequencing Assay, Plasmid Preparation, shRNA, Mutagenesis, Generated, Clone Assay, Software, Microarray