p130cas knockdown (Thermo Fisher)
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Thermo Fisher
p130cas knockdown
P130cas Knockdown, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/p130cas knockdown/product/Thermo Fisher
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
P130cas Knockdown, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/p130cas knockdown/product/Thermo Fisher
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
p130cas knockdown - by Bioz Stars,
2024-12
86/100 stars
Images
1) Product Images from "Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation"
Article Title: Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation
Journal: bioRxiv
doi: 10.1101/2023.11.22.568289
Figure Legend Snippet: (A) Plot of p130Cas ( Homo sapiens ) amino-acid position versus predicted disorder probability using web-based tool Protein DisOrder (PrDOS). Red indicates high probability of disorder. Top: Color-coded protein domains. Horizontal line at 0.5 represents 5% false positive rate prediction. (B) Time-lapse images showing the emergence of a p130Cas droplet from a focal adhesion (FA) within a live NIH3T3 cell. Intensity scale bar to the right; black/blue represents low intensity while white/yellow represents high intensity. Corresponding intensity line profiles perpendicular to the FA long axis are shown in the lower panels. Arrow (white) and line profile (black) indicates droplet formation. Note that the p130Cas intensity in the droplet is significantly higher than in the cytoplasm or FAs. Scale bar = 10µm. (C) Time-lapse imaging shows coalescence of two droplets in a live NIH3T3 cell; corresponding intensity line scan in the lower panel. Scale bar = 10µm. (D) Histogram of p130Cas droplet area at ∼6 hr (black) and ∼24 hr (red) after plating transfected NIH3T3 fibroblasts on fibronectin-coated glass bottom dishes. N = 521 droplets from 48 cells and N = 631 droplets from 121 cells for 6 hr and 24 hr respectively. Inset: Mean droplet area at 6 hr and 24 hr. Error bars = standard error of the mean (SEM). p-value<5×10 -6 using Student’s t-test. (E-F) Time lapse images showing droplet intensity (upper panel) and quantified intensity profile (lower panel) during FRAP at pre-bleach (-2s), immediately after photobleach (0s) and during recovery in cells at 6 hr (E) and 24 hr (F) after plating. Scale bar = 5µm (E) & 10µm (F). (G-I) Plot of normalized fluorescence intensity or recovery fraction with time for droplets at 6 hr (black curve) and 24 hr (red curve) and the corresponding mean mobile fraction (H) and t 1/2 for recovery (I) determined by fitting individual FRAP curves to single component exponential recovery function. N = 14 for 6 hr and N = 7 for 24 hr. Error bars = standard deviation (SD) in (G) and SEM in (H-I). (J-K) Intensity coded image (J) and quantification of number of droplets (K) (N = 21 cells) before and after treatment with 5% hexanediol for 2 min. Error bars are standard deviations. Scale bar = 50µm. (L-M) Intensity coded image (L) and quantification of number of droplets (M) (N = 11 cells) before and after treatment with 100mM ammonium acetate for 8min. Scale bar = 50µm. Error bars = SD.
Techniques Used: Imaging, Transfection, Fluorescence, Standard Deviation
Figure Legend Snippet: (A) Comparison of disorder probability of Homo sapiens (human) and Mus musculus (mouse) p130Cas. Plot of disorder probability prediction versus amino-acid residue position in human (black) and mouse (red) p130Cas/BCAR1. Top: Color-coded protein domains. Horizontal line at 0.5 represents 5% false positive rate prediction. (B) Immunoblot of p130Cas in EGFP-p130Cas NIH3T3 cell line under transient over-expression. (C) Immunofluorescence image of transiently transfected NIH3T3 with EGFP-p130Cas (green) stained with anti-p130Cas antibody (red) showing colocalization. Arrows points to droplets. Scale bar = 20µm. (D-F) Inverted intensity images of EGFP-p130Cas transfected in (D) Chinese Hamster Ovary (CHO) cells (Scale bar = 50µm), (E) HeLa cells (Scale bar = 20µm) and (F) HEK 293Tx cells (Scale bar = 20µm). (G) Immunoblot of p130Cas in NIH3T3 and MCF7 cell line with GAPDH as a loading control. MCF7 cells express 2-2.5x more p130Cas than NIH3T3 fibroblasts. (H) Inverted intensity immunofluorescence image of endogenous p130Cas in MCF7 cells stained with anti-p130Cas antibody. Scale bar = 20µm.
Techniques Used: Comparison, Residue, Western Blot, Over Expression, Immunofluorescence, Transfection, Staining, Control
Figure Legend Snippet: (A & C) EGFP-p130Cas (green) transfected cells immunostained for paxillin (red) (A) and FAK (C). Lower panels show zoomed-in images of the area in the white box. (B & D) Line scans of p130Cas and paxillin (B) / FAK (D) showing co-localization. (E & F) Time lapse images of a cell co-transfected with EGFP-p130Cas (green) and either tagRFP-paxillin (red) (E) or mcherry-FAK (red) (F) showing the co-emergence from a FA of paxillin (E) and FAK (F) with p130Cas. Lower panel: corresponding line intensity profile. (G & H) EGFP-p130Cas in a paxillin-null (G) and FAK-null (H) cell. Right panels show zoomed-in images of the boxed area. (I & J) Image of a p130Cas-null cell transfected with either tagRFP-paxillin (I) or mcherry-FAK (J). Right panels show zoomed-in images of the boxed area. (K) Immunofluorescence image of a cell expressing EGFP-p130Cas (green) stained for phosphorylated p130Cas (purple). (L) Lower panels show zoomed-in images of the boxed area. (M) Corresponding intensity line profile of phosphorylated p130Cas and p130Cas. (N) Ratio of phosphorylated p130Cas to total p130Cas) in FA and droplets. N = 2176 FA and 53 droplets from 10 cells each. Error bars = SEM. (O) Schematic of WT, Y15F mutant, substrate domain deleted (Δ68-456 or ΔSD) and C-terminal domain partly deleted p130Cas (Δ611-742 or C-term). (P) Image of p130Cas-/- cells transfected with EGFP-WT-, Y15F, ΔSD/ Δ68-456 or C-term/ Δ611-742 p130Cas. (Q) Quantification of percentage of droplet-positive cells for the indicated constructs from N = 7 (N indicates independent experiments) with 146/238, 76/156, 231/347, 56/86, 205/396, 236/430 and 110/284 cells; N = 4 with 82/278, 52/201, 42/184 and 58/238 cells; N = 3 with 38/346, 23/203 and 68/371 cells and N = 3 with 139/287, 142/222 and 134/325 cells respectively. Error bars = SEM. (R) Plot of disorder probability versus amino acid for WT (black) and Y15F mutant (red) mouse p130Cas.Top: Color-coded protein domains.
Techniques Used: Transfection, Immunofluorescence, Expressing, Staining, Mutagenesis, Construct
Figure Legend Snippet: (A) Cell co-transfected with EGFP-p130Cas (green-left panel) and tagRFP-paxillin (red-middle panel) showing colocalization (merge-right panel). (B-C) Corresponding line intensity profiles for a typical droplet (B) and a FA (C). (D-E) Cell co-transfected with EGFP-p130Cas (green-left panel) and tagRFP-paxillin (D) or mCherry-FAK (E) (red-middle panel) showing colocalization in droplets with (lower panel) or without (upper-panel-control)) 250mM ammonium acetate treatment.
Techniques Used: Transfection, Control
Figure Legend Snippet: (A & B) Fluorescence image of GFP-trap magnetic beads incubated with cell lysate from HEK293tx cells transfected with either EGFP-p130Cas (A) or EGFP (B) and then mixed with lysate from tagRFP-paxillin transfected cells. Images show p130Cas on beads (green-left panel), paxillin on beads (red-middle panel) and DIC images of beads (gray-right panel): First row-Untreated, second row- 250mM ammonium acetate, third row- 5% hexanediol, fourth row-washed with PBS. (C & D) GFP-trap beads incubated with cell lysate from HEK293tx cells transfected with either EGFP-p130Cas (C) or EGFP (negative control) (D) plus lysate from tagRFP-paxillin cells. Beads treated as in A,B were fixed with 4% paraformaldehyde and then washed with cold PBS. Note retention of paxillin after washing. (E) Proteins specifically associated with p130Cas beads (≥ 2 counts and 2-fold enrichment compared with control beads) were analyzed for Gene Ontology (GO; biological processes) using the online webtool-Database for Annotation, Visualization, and Integrated Discovery (DAVID). Number of proteins versus corresponding GO term is plotted with fold enrichment depicted by the size of the circle and -log10(P-value) by the color of the circle. The corresponding fold enrichment and -log10(P-value) scale bars are shown to the right of each plot. GO-terms are sorted in descending order of their -log10(P-values).
Techniques Used: Fluorescence, Magnetic Beads, Incubation, Transfection, Negative Control, Control
Figure Legend Snippet: Venn diagram showing the number of proteins significantly enriched in p130Cas droplets relative to GFP-only vs its published direct interactors.
Techniques Used:
Figure Legend Snippet: (A, C, E & G) Cells expressing EGFP-p130Cas (red-left panel) stained for G3BP2 (A & C), PABP (E) or Ago2 (G) (green - middle panel) and merged (right panel) under control/unstimulated (A) and stress granule induction with sodium arsenite (C, E & G). (B, D, F & H) The corresponding line intensity profile across a p130Cas droplet and a stress granule. (I) Immunofluorescence image of p130Cas (red - middle panel) in HEK293T cells stably expressing EGFP-Dcp1A marking p-granules (green-left panel) and the merge (right panel). (J) The corresponding line intensity profile across a p130Cas droplet and a p-granule. (K) Immunofluorescence image of Dcp1A (green - middle panel) in NIH3T3 cells expressing tagRFP-p130Cas (red-left panel) and the merge (right panel). (L) The corresponding line intensity profile across a p130Cas droplet and a p-granule.
Techniques Used: Expressing, Staining, Control, Immunofluorescence, Stable Transfection
Figure Legend Snippet: (A) HEK293T cells stably expressing EGFP-Dcp1A (left-panel) transfected with tagRFP-p130Cas (red-middle panel) and merged (right-panel). (B) A typical line intensity profile across a p130Cas droplet and a p granule.
Techniques Used: Stable Transfection, Expressing, Transfection
Figure Legend Snippet: (A & C) Cells expressing EGFP-p130Cas (green-left panel) stained for Ago2 (A) and GW182 (C) (purple-middle panel) and merged (right panel). (B & D) Corresponding line intensity profile across a p130Cas droplet showing its colocalization with Ago2 present throughout the droplet (A) and GW182 at the periphery of the droplet (C). (E) Cells expressing tagRFP-p130Cas (red-left panel) with RNA-fluorescence in-situ hybridization (FISH) of poly-A binding probes (green-middle panel) and merged. (F) The corresponding line intensity profile across a p130Cas droplet showing colocalization with RNA-FISH probes. (G) EGFP-p130Cas expressing cells plated for 5hr without (-CHX) or with (+CHX) 100µg/ml cycloheximide for 2 minutes. (H) Number of droplets per cell under these conditions. N = 3 independent experiments with 39 and 52 cells for -CHX and +CHX respectively. Bar represents Mean, horizontal line is median and Error bars = SD.
Techniques Used: Expressing, Staining, Fluorescence, In Situ Hybridization, Binding Assay
Figure Legend Snippet: Immunofluorescence of Ago2 (A & B) and GW182 (C & D) (purple-middle panel) on GFP-Trap magnetic beads incubated with cell lysate from HEK293tx cells transfected with either EGFP-p130Cas (A & C) or EGFP (B & D) after fixation with 4% PFA. Corresponding DIC images, right panel.
Techniques Used: Immunofluorescence, Magnetic Beads, Incubation, Transfection
Figure Legend Snippet: (A) Volcano plot of mRNAs enriched on p130Cas beads compared to the EGFP-only control. mRNAs with 2-fold enrichment and p-value<0.05 are associated with p130Cas droplet (red dots). (B) Gene Ontology (GO) analysis (biological processes) of p130Cas-enriched mRNAs as described in Methods. (C) Volcano plot of p130Cas-regulated mRNAs. mRNAs with 2-fold up-regulation (red dots) or down-regulation (green dots) after p130Cas expression compared to the control transfected. (D) Gene Ontology (GO) analysis (biological processes) of p130Cas rgulated mRNAs as described in Methods. Number of mRNAs versus corresponding GO term is plotted with fold enrichment depicted by the size of the circle and -log10(P-value) by the color of the circle. The corresponding fold enrichment and -log10(P-value) scale bars are shown to the right of each plot. GO-terms are sorted in the descending order of the mRNA count (B) or -log10(P-values) (D).
Techniques Used: Control, Expressing, Transfection
Figure Legend Snippet: (A-B) MCF7 on low (2µg/ml), medium (20µg/ml) and high (50µg/ml) fibronectin-coated dishes stained for paxillin (red) with the nucleus labelled using Hoechst-33343. Zoomed-in images (B) of the area labelled in the white box in (A) showing punctate paxillin in the cytosol to mark droplets. Scale bar = 20µm (A) and 5µm (B). (C) Quantification of puncta in MCF7 cells from A&B. N = 5 field of views (FOVs) with 3-6 cells per field of view from 3 independent experiments imaged using high resolution 60× objective. (D) Intensity-coded images of MCF7 cells on low, medium, and high FN incubated with puromycin then fixed and stained with anti-puromycin antibody. Scale bar = 100µm. All images in a panel are shown at same intensity scale as depicted in the right color bar with black/blue as low intensity and white/yellow as high intensity. (E) Quantification of puromycin labelling intensity from D. N = 262, 285 and 240 cells from 16 field of views each for low, medium, and high fibronectin respectively from 3 independent experiments. (F & I) Intensity-coded images of puromycin labelling in MCF7 cells (F) & HUVECs (I) transfected with scrambled siRNAs or p130Cas siRNA on low and high fibronectin coated substrate. Scale bar = 100µm. (G & J) Immunoblot of p130Cas for cells in (G) and HUVECs (J), with tubulin (G) and GAPDH (J) as loading controls. (H & K) Quantification of puromycin intensity for multiple cells MCF7 from F & I. N = 690, 331, 547 & 359 MCF7 cells and N = 792, 873, 1232 & 1054 HUVECs from 25 FOVs each from 3 independent experiments for cells on low and high fibronectin without and with p130Cas knock down respectively. (L) Intensity coded images of puromycin labelling in MCF10A cells plated on low and high fibronectin coated substrate. Scale bar = 50µm. (M) Quantification of puromycin intensity from multiple cells from L. N = 859 and 863 cells from 25 field of views from 3 independent experiment for cells on low and high fibronectin respectively.
Techniques Used: Staining, Incubation, Transfection, Western Blot, Knockdown
Figure Legend Snippet: (A) NIH 3T3 cells on low or high FN labeled with puromycin (purple), nucleus labeled with hoeschst 33343. Cells were treated with or without 100µg/ml cycloheximide for 2 min before puromycin labelling for 10 min. (B) Quantification of p130Cas Western blot after knock-down in MCF7 cells. (C) HUVECs on low vs high FN labeled with puromycin as in A. (D) Quantification of puromycin labelling intensity in C. N = 388 and 348 cells from 9 field of views each from 3 independent experiments for low and high FN conditions respectively. (E) NIH 3T3 cells transfected with control or p130Cas siRNA on low vs. high FN, labeled for puromycin as in (A). (F) Immunoblot of p130Cas for NIH 3T3 cells in (E), GAPDH as loading controls. Labelling intensity quantified in (G). N = 4 field of views each from 4 independent experiments for each condition.
Techniques Used: Labeling, Western Blot, Knockdown, Transfection, Control
Figure Legend Snippet: (A-B) MCF7 cells expressing light inducible cry2-tagRFP-p130Cas illuminated with blue LED light (red-left panel) stained for paxillin (A) or FAK (B) (green-middle panel) and merged (right panel). Scale bar = 5µm. (C) Intensity coded images of puromycin labelling in stable MCF7 cell line with modest over-expression of cry2-tagRFP-p130Cas plated on low and high fibronectin coated substrate. Scale bar = 100µm. (D) Quantification of puromycin intensity in multiple cells from C. N = 446 and 747 cells from 25 field of views for cells on low and high fibronectin respectively from 3 independent experiments. (E) Time lapse intensity inverted images of MCF7 cell stably expressing cry2-tagRFP-p130Cas before (first frame) and after (second frame onwards) a 0.5 sec 488nm laser pulse. Scale bar = 50µm. Lower panels show zoomed-in images of the area labelled in the red box. Scale bar = 20µm. (F) Typical plot of the droplets per cell over time after illumination of cry2-tagRFP-p130Cas cells. Red line shows the fit to an exponential function to determine the half-life of the droplets. Inset: Box plot of half-life of p130Cas droplets showing mean at 9.5 ± 3.7 minutes. N = 8 cells. (G) Intensity inverted images of cry2-tagRFP-p130Cas cell line with no blue LED light induction (first image) and cells illuminated with pulsed blue LED light for 2hr then incubated in the dark for indicated times. Cells were then pulsed with puromycin, fixed and stained. Scale bar = 100µm. (H-I) Quantification of droplet area percentage (H) and puromycin labelling intensity (I) in cells from (G). N > 550 cells from 25 FOVS from 3 independent experiments each for No LED light, 0, 11, 15 and 25 minutes respectively after switching off the blue LED after intermittently illumination for 2hr. (J) Intensity inverted images of cry2-tagRFP-p130Cas cell line with no light (first image) or illuminated for with pulsed LED light for the indicated times, labelled with puromycin and stained. Scale bar = 100µm. (K-L) Quantification of droplet area % (K) and puromycin labelling intensity (L) for cells in (J). N > 550 cells from 25 FOVS from 3 independent experiments each for No blue LED light or LED switched on for 11, 15, 25 and 40 minutes respectively with intermittent pulses.
Techniques Used: Expressing, Staining, Over Expression, Stable Transfection, Incubation
Figure Legend Snippet: (A-B) MCF7 cells expressing light-inducible cry2-tagRFP-p130Cas after exposure to 470nm LED light and stained for paxillin (A) or FAK (B) (green-middle panel) in (red-left panel), merge-right panel. Scale bar = 50µm. (C) MCF7 cells stably expressing cry2-tagRFP-p130Cas were sorted for modest vs high expressors and cells analyzed for tagRFP intensity. Top panel: untransfected MCF7 cells (negative control), unsorted cells (middle panel), sorted cells (modest over-expression -light blue) (bottom panel). (D) Immunoblot of p130Cas in WT and stable cry2-tagRFP-p130Cas expressing MCF7 cell line with GAPDH as a loading control. (E) Pulse sequence with 5 sec ON followed by 60 sec OFF to minimize phototoxicity. Blue LED light array was controlled using programmable switch-Arduinos. (F). Puromycin labelled control WT MCF7 cells after 0- and 120-min exposure to blue LED light pulse protocol and (G) quantification of normalized puromycin intensity under these two conditions showing no effect of illumination on protein translation. N = 680 and 591 cells respectively for 0 and 120 min from 16 field of views each from 3 independent experiments.
Techniques Used: Expressing, Staining, Stable Transfection, Negative Control, Over Expression, Western Blot, Control, Sequencing
