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mouse anti-jnk2 antibody (Santa Cruz Biotechnology)

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Santa Cruz Biotechnology mouse anti-jnk2 antibody
Mouse Anti Jnk2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+anti+jnk2/anti+c+jun/us12227770-445-0-3
Average 90 stars, based on 1 article reviews

mouse anti-jnk2 antibody - by Bioz Stars, 2026-09

90/100 stars

Images

Incubation:

Article Title: JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.
Article Snippet: .. Triton X-100, 15% [vol./vol.] donkey serum in PBS) for 1 h. Primary antibodies were incubated overnight at 4°C after dilution in 1.5% (vol./vol.) donkey serum at the following concentrations: 1:100 rabbit anti-JNK3 (Upstate, Lake Placid, NY, USA), 1:50 mouse anti-JNK2 (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) and 1:500 guinea pig anti-insulin (Dako, Carpinteria, CA, USA). .. After extensive washes in PBS, the sections were incubated for 2 h at room temperature in the appropriate secondary biotinylated donkey anti-mouse or donkey antirabbit or donkey anti-guinea pig antibody (Jackson Immunoresearch, West Grove, PA, USA) diluted 1:500 in PBS.

Western Blot:

Article Title: Constitutive Activity of JNK2?2 Is Dependent on a Unique Mechanism of MAPK Activation
Article Snippet: .. The following antibodies were used for immunoblotting: mouse anti-GFP (catalog number 11814460001; Roche Applied Science), rabbit anti-phosphorylated JNK that recognizes the phosphorylated T-P-Y motif (98F2; Cell Signaling Technology), rabbit anti-phosphorylated c-Jun (9261; Cell Signaling Technology), mouse anti-JNK2 (sc-7345; Santa Cruz), mouse anti-actin (MAB1501R; Chemicon), mouse ant-GST (sc-138; Santa Cruz), and mouse anti-FLAG (Sigma; F3165). .. Immunoprecipitations for 3×FLAG constructs were conducted in radioimmunoprecipitation assay buffer using 2 μg of mouse anti-FLAG antibody (F3165; Sigma) bound to Gammabind G-Sepharose (GE Healthcare).

Article Title: Constitutive Activity of JNK2α2 Is Dependent on a Unique Mechanism of MAPK Activation
Article Snippet: .. The following antibodies were used for immunoblotting: mouse anti-GFP (catalog number 11814460001; RocheApplied Science), rabbit anti-phosphorylated JNK that recognizes the phosphorylated T-P-Y motif (98F2; Cell Signaling Technology), rabbit anti-phosphorylated c-Jun (9261; Cell Signaling Technology), mouse anti-JNK2 (sc-7345; Santa Cruz), mouse anti-actin (MAB1501R; Chemicon), mouse ant-GST (sc-138; Santa Cruz), and mouse anti-FLAG (Sigma; F3165). .. Immunoprecipitations for 3 FLAG constructs were conducted in radioimmunoprecipitation assay buffer using 2 g of mouse anti-FLAG antibody (F3165; Sigma) bound to Gammabind G-Sepharose (GE Healthcare).

Article Title: Angiogenic growth factors inhibit chondrocyte ageing in osteoarthritis: potential involvement of catabolic stress-induced overexpression of caveolin-1 in cellular ageing
Article Snippet: Objective: Recently, attention has been attracted by the finding that overexpression of caveolin-1 induces cellular senescence in age-related diseases.. We aimed to ascertain whether angiogenic growth factors (AGFs) can inhibit interleukin (IL)-1β-induced senescence in human chondrocytes by downregulation of caveolin-1.. Methods: We investigated the intracellular signalling pathways involved in chondrocyte ageing.

Activity Assay:

Article Title: Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.
Article Snippet: .. Antibodies: goat anti-mouse antibody coupled to alkaline phosphatase (goat polyclonal, sc-2008), goat anti-rabbit antibody coupled to alkaline phosphatase (goat polyclonal, sc-2007), mouse anti-ubiquinolcytochrome c reductase core protein I (UQCRC1) (16D10 mAb, IgG1, sc-65238), mouse anti-NF-kB p65 (F-6 mAb, IgG1, sc-8008), mouse anti-IkB-a (H-4 mAb, IgG1, sc-1643), mouse anti p-p38 (D-8 mAb, IgM, sc-7973), mouse anti-p38a/b (A-12 mAb, IgG1, sc-7972), mouse anti-histone H1 (AE-4 mAb, IgG2a, sc-8030), and mouse anti-JNK2 (A-7 mAb IgG1, sc-271133) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); mouse anti-IL-1b (mAb, IgG1, I3642; the antibody neutralizes the biological activity of recombinant human IL-1b) and rabbit anti-actin antibody (polyclonal, A2066) were from Sigma-Aldrich. .. Cell Cultures IB3-1 cells (ATCC CRL-2777, a bronchial cell line derived from a cystic fibrosis patient with a DF508/W1282X CFTR genotype) [56] and S9 cells (ATCC CRL-2778, which are IB3-1 cells transduced with an adeno-associated viral vector to stably express wt-CFTR) [57] were purchased from ATCC (www.atcc. org) (these cell lines are no longer provided by ATCC; they are kept now at the John Hopkins University Cell Center).

Article Title: Disruption of Interleukin-1β Autocrine Signaling Rescues Complex I Activity and Improves ROS Levels in Immortalized Epithelial Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function
Article Snippet: .. Antibodies: goat anti-mouse antibody coupled to alkaline phosphatase (goat polyclonal, sc-2008), goat anti-rabbit antibody coupled to alkaline phosphatase (goat polyclonal, sc-2007), mouse anti-ubiquinol-cytochrome c reductase core protein I (UQCRC1) (16D10 mAb, IgG 1 , sc-65238), mouse anti-NF-κB p65 (F-6 mAb, IgG 1 , sc-8008), mouse anti-IκB-α (H-4 mAb, IgG 1 , sc-1643), mouse anti p-p38 (D-8 mAb, IgM, sc-7973), mouse anti-p38α/β (A-12 mAb, IgG 1 , sc-7972), mouse anti-histone H1 (AE-4 mAb, IgG 2a , sc-8030), and mouse anti-JNK2 (A-7 mAb IgG 1 , sc-271133) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); mouse anti-IL-1β (mAb, IgG 1 , I3642; the antibody neutralizes the biological activity of recombinant human IL-1β) and rabbit anti-actin antibody (polyclonal, A2066) were from Sigma-Aldrich. .. IB3-1 cells (ATCC CRL-2777, a bronchial cell line derived from a cystic fibrosis patient with a ΔF508/W1282X CFTR genotype) and S9 cells (ATCC CRL-2778, which are IB3-1 cells transduced with an adeno-associated viral vector to stably express wt-CFTR) were purchased from ATCC ( www.atcc.org ) (these cell lines are no longer provided by ATCC; they are kept now at the John Hopkins University Cell Center).

Recombinant:

Membrane:

Image Search Results

Proteomic analysis of Tg2576 mice infused with blood from old and young wild type mice. ( A ) Heatmap of quantifiable proteins from the Old Blood and Young Blood groups, showing log fold-change (logFC) in protein expression. ( B ) Venn diagram of differentially expressed proteins in an Old Blood vs. Young Blood groups comparison. ( C ) Canonical pathway analysis of differentially expressed proteins, highlighting enriched signaling pathways with activation z-scores. ( D – F ) Differentially expressed proteins in cAMP-mediated signaling, synaptogenesis signaling, and endocannabinoid neuronal synapse pathways, respectively. Gene names are used. ( G , H ) Representative western blot image and quantitative analysis of expression of CACNA2D2 in brain homogenates. ( I , J ) Representative western blot image and quantitative analysis of expression of BRAF in brain homogenates. ( K , L ) Representative western blot image and quantitative analysis of expression of Syngap1 in brain homogenates. ( M , N ) Representative western blot image and quantitative analysis of expression of MAPK9 in brain homogenates. ( O , P ) Representative western blot image and quantitative analysis of expression of GRK2 in brain homogenates. N = 3/group for proteomic analysis, and n = 4/group for protein validation (random mix of males and females; young donor group: 1–2M/2F; old donor group: 1–2M/2F). Data are expressed as mean ± SEM. Data in ( H ), ( J ), ( L ), ( N ), and ( P ) were analyzed using Student’s t -test. * p < 0.05.

Journal: Aging (Albany NY)

Article Title: Infusion of blood from young and old mice modulates amyloid pathology

doi: 10.18632/aging.206319

Figure Lengend Snippet: Proteomic analysis of Tg2576 mice infused with blood from old and young wild type mice. ( A ) Heatmap of quantifiable proteins from the Old Blood and Young Blood groups, showing log fold-change (logFC) in protein expression. ( B ) Venn diagram of differentially expressed proteins in an Old Blood vs. Young Blood groups comparison. ( C ) Canonical pathway analysis of differentially expressed proteins, highlighting enriched signaling pathways with activation z-scores. ( D – F ) Differentially expressed proteins in cAMP-mediated signaling, synaptogenesis signaling, and endocannabinoid neuronal synapse pathways, respectively. Gene names are used. ( G , H ) Representative western blot image and quantitative analysis of expression of CACNA2D2 in brain homogenates. ( I , J ) Representative western blot image and quantitative analysis of expression of BRAF in brain homogenates. ( K , L ) Representative western blot image and quantitative analysis of expression of Syngap1 in brain homogenates. ( M , N ) Representative western blot image and quantitative analysis of expression of MAPK9 in brain homogenates. ( O , P ) Representative western blot image and quantitative analysis of expression of GRK2 in brain homogenates. N = 3/group for proteomic analysis, and n = 4/group for protein validation (random mix of males and females; young donor group: 1–2M/2F; old donor group: 1–2M/2F). Data are expressed as mean ± SEM. Data in ( H ), ( J ), ( L ), ( N ), and ( P ) were analyzed using Student’s t -test. * p < 0.05.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4°C in agitation: 6E10 mouse/IgG1 (Biolegend, San Diego, CA, USA) (1/500), α2δ2 ( Cacna2d2 ) rabbit/IgG (Abcam, Fremont, CA, USA) (1:1000), SynGAP1 rabbit/IgG (Abcam, Fremont, CA, USA), BRAF rabbit/IgG (Proteintech, Rosemont, IL, USA), JNK2 (MAPK9) mouse/IgG (Origene, Rockville, MD, USA), GRK2 mouse/IgG1 (Invitrogen, Carlsbad, CA, USA), and β-Actin mouse/IgG2b (Cellsignal, Danvers, MA, USA).

Techniques: Expressing, Comparison, Protein-Protein interactions, Activation Assay, Western Blot, Biomarker Discovery

FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for 5 min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in . (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for integrin αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from . N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F (5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F (9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for 5 min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in . (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for integrin αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from . N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F (5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F (9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: anti-human integrin β1 clones 12G10 (NB100-63255; Novus bio), mAb13 (552828; BD), total integrin β1 (MAB2252; Millipore), anti-human integrin αvβ5 clone 15F11 (MAB2019Z; Millipore), anti-human integrin α5 clone SNAKA51 (AF1846; R&D), anti-human Tensin1 (SAB4200283; Sigma-Aldrich), anti-human p-paxillin Y118 (69363; Cell Signaling), anti-human-Talin1 (T3287; Sigma-Aldrich), anti-α-tubulin (sc-32293; SantaCruz Biotechnology), and anti-GAPDH (G9545; Sigma-Aldrich).

Techniques: Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction

FN inhibits RA formation in a local manner. (A) U2Os-AP2-GFP cells plated on FN, VTN, Col I, Col IV, LN111 (all 10 µg/ml) -coated or non-coated dishes, stained for p-Pax and integrin αvβ. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N = 21 from two independent experiments. F (5, 120) = 48.05, P < 0.0001. (C) U2Os-AP2-GFP cells were plated on FN/glass patterned imaging dishes overnight, stained for integrin αvβ5 and p-Pax, and imaged using TIRF microscopy; representative TIRF images. (D) Quantification of integrin αvβ5 fluorescent intensity on FN- and glass-side of the pattern; n = 10 from one representative experiment. Similar results were observed in five similar experiments. Two-tailed Student’s t test, P < 0.01. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FN inhibits RA formation in a local manner. (A) U2Os-AP2-GFP cells plated on FN, VTN, Col I, Col IV, LN111 (all 10 µg/ml) -coated or non-coated dishes, stained for p-Pax and integrin αvβ. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N = 21 from two independent experiments. F (5, 120) = 48.05, P < 0.0001. (C) U2Os-AP2-GFP cells were plated on FN/glass patterned imaging dishes overnight, stained for integrin αvβ5 and p-Pax, and imaged using TIRF microscopy; representative TIRF images. (D) Quantification of integrin αvβ5 fluorescent intensity on FN- and glass-side of the pattern; n = 10 from one representative experiment. Similar results were observed in five similar experiments. Two-tailed Student’s t test, P < 0.01. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Techniques: Staining, Imaging, Microscopy, Two Tailed Test

FCLs and RAs presence correlates with FN production in multiple cells lines. (A–E) The following knock-in cell lines were used in this figure: U2Os-AP2-GFP, HeLa-AP2-GFP, hMEC-AP2-GFP, U2Os-AP2-halo, HeLa-AP2-halo, MCF7-AP2-halo, HDF-AP2-halo, and Caco2-AP2-halo. (A) Cell lines indicated were plated to non-coated (nc) dishes, allowed to settle overnight, and stained for integrin αvβ5 and FN. Representative TIRF images. (B) Analysis of FN fluorescent intensity from samples in A. N (images): U2Os = 21, HeLa = 20, MCF7 = 23, HDF = 33, Caco2 = 16, hMEC = 23, from two independent experiments. F (5, 130) = 56.16, P < 0.0001. (C) Cells were plated on FN-coated or nc dishes, allowed to settle overnight, and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C. Cell lines with an AP2 halo tag, n (images): U2Os (nc/FN) = 20, HeLa (nc) = 33, HeLa (FN) = 29, MCF7 (nc) = 29, MCF7 (FN) = 32, HDF (nc/FN) 32, Caco2 (nc/FN) = 32. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (9, 273) = 64.96, P < 0.0001. (E) Analysis of RA coverage from samples in C. Cell lines with an AP2 GFP tag, n (images): U2Os (nc) = 31, U2Os (FN) = 18, HeLa (nc) = 35, HeLa (FN) = 38, and hMEC (nc/FN) = 38. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (5, 189) = 89.97, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: FCLs and RAs presence correlates with FN production in multiple cells lines. (A–E) The following knock-in cell lines were used in this figure: U2Os-AP2-GFP, HeLa-AP2-GFP, hMEC-AP2-GFP, U2Os-AP2-halo, HeLa-AP2-halo, MCF7-AP2-halo, HDF-AP2-halo, and Caco2-AP2-halo. (A) Cell lines indicated were plated to non-coated (nc) dishes, allowed to settle overnight, and stained for integrin αvβ5 and FN. Representative TIRF images. (B) Analysis of FN fluorescent intensity from samples in A. N (images): U2Os = 21, HeLa = 20, MCF7 = 23, HDF = 33, Caco2 = 16, hMEC = 23, from two independent experiments. F (5, 130) = 56.16, P < 0.0001. (C) Cells were plated on FN-coated or nc dishes, allowed to settle overnight, and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C. Cell lines with an AP2 halo tag, n (images): U2Os (nc/FN) = 20, HeLa (nc) = 33, HeLa (FN) = 29, MCF7 (nc) = 29, MCF7 (FN) = 32, HDF (nc/FN) 32, Caco2 (nc/FN) = 32. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (9, 273) = 64.96, P < 0.0001. (E) Analysis of RA coverage from samples in C. Cell lines with an AP2 GFP tag, n (images): U2Os (nc) = 31, U2Os (FN) = 18, HeLa (nc) = 35, HeLa (FN) = 38, and hMEC (nc/FN) = 38. Data were obtained from two individual experiments and similar results were observed in four individual experiments. F (5, 189) = 89.97, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 µm.

Techniques: Knock-In, Staining

Integrin αvβ5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin αvβ5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin αvβ5 and p-Pax. Representative TIRF images. (E) Analysis of integrin β5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin αvβ5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin αvβ5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F (2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P -value; *** P -value < 0.001. Scale bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Integrin αvβ5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin αvβ5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin αvβ5 and p-Pax. Representative TIRF images. (E) Analysis of integrin β5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin αvβ5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin αvβ5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F (2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P -value; *** P -value < 0.001. Scale bars, 10 µm.

Techniques: Staining, Control, Microscopy, Comparison

Inhibition of CME prevents RA formation. (A) U2Os-AP2-GFP cells silenced for AP2A1, AP2S1, or control (scrambled shRNA) were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (5, 64) = 43.11, P < 0.001. (C) U2Os-AP2-GFP cells overexpressing Ap180 ct were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C; n = 17 from one representative experiment and similar results were observed in four individual experiments. Two-tailed Student’s t test, P < 0.0001. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Inhibition of CME prevents RA formation. (A) U2Os-AP2-GFP cells silenced for AP2A1, AP2S1, or control (scrambled shRNA) were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of RA coverage from samples in A. N (images): control = 12, shAP2A1#1 = 8, shAPA1#2 = 11, shAPS1#1 = 10, shAPS1#2 = 19, shAPS1#3 = 10. Data were obtained from two individual experiments and similar results were observed in five independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (5, 64) = 43.11, P < 0.001. (C) U2Os-AP2-GFP cells overexpressing Ap180 ct were plated on non-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from samples in C; n = 17 from one representative experiment and similar results were observed in four individual experiments. Two-tailed Student’s t test, P < 0.0001. (E) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Techniques: Inhibition, Control, shRNA, Staining, Comparison, Two Tailed Test

RAs are formed at FCLs. (A) U2Os-AP2-ITGB5-mScarlet cells plated on glass were treated with Cilengitide (10 µM) for 15 min, washed twice with fresh medium, and imaged live using TIRF microscopy at two frames per minute. Representative frames showing the growth of an RA from an FCL. (B) Analysis of AP2 and integrin αvβ5 (ITGB5) intensity over time. Left: FCL and αvβ5 clustering during RA assembly ( n = 16 events). In this graph, time zero is defined as the arrival of the AP2 signal Right: ITGB5 clusters not stabilized by FCLs disassemble and are removed from the membrane by CME-mediated disassembly ( n = 24 events). In this graph, time zero is defined as the disappearance of the ITGB5 signal. Events have been collected from four 1-h long time-lapse acquisitions. Mean ± SEM. (C) A schematic illustration of the results is shown in this figure. Scale bar, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: RAs are formed at FCLs. (A) U2Os-AP2-ITGB5-mScarlet cells plated on glass were treated with Cilengitide (10 µM) for 15 min, washed twice with fresh medium, and imaged live using TIRF microscopy at two frames per minute. Representative frames showing the growth of an RA from an FCL. (B) Analysis of AP2 and integrin αvβ5 (ITGB5) intensity over time. Left: FCL and αvβ5 clustering during RA assembly ( n = 16 events). In this graph, time zero is defined as the arrival of the AP2 signal Right: ITGB5 clusters not stabilized by FCLs disassemble and are removed from the membrane by CME-mediated disassembly ( n = 24 events). In this graph, time zero is defined as the disappearance of the ITGB5 signal. Events have been collected from four 1-h long time-lapse acquisitions. Mean ± SEM. (C) A schematic illustration of the results is shown in this figure. Scale bar, 5 µm.

Techniques: Microscopy, Membrane

$Integrin β1 blocking stimulates FCL and RA formation. (A) U2Os-AP2-GFP cells plated on FN were treated with integrin β1 blocking antibody mab13 (0.3 µg/ml) for 15 and 45 min (or vehicle for 45 min = control) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of AP2 signal colocalizing with no markers (non), p-Pax, or integrin αvβ5 over time. N (images): control = 18, mab13 15 min = 13, mab13 45 min = 15, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 119) = 46.68, P < 0.0001. (C) Analysis of fraction of integrin αvβ5 colocalizing with AP2 over time from samples in A. N (images): control = 15, mab13 15 min = 11, mab13 45 min = 12, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 35) = 45.81, P < 0.0001. (D) U2Os-AP2-GFP cells plated on FN were treated with mab13 (0.3 µg/ml) and imaged using TIRF microscopy. 5-min time-lapses with 1-s intervals starting at 0 min (no mab13) and every 5 min after mab13 addition, until 35 min, were acquired. N (videos): 0 min = 11, 5 min = 11, 10 min = 9, 15 min = 8, 20 min = 8, 25 min = 6, 30 min = 7, 35 min = 6. Videos were acquired from two independent experiments. Similar results were observed in five individual experiments. One-way ANOVA with Tukey’s multiple comparison. F (7, 29) = 8.893, P < 0.0001. (E) A schematic illustration of results shown in this figure. Data are the mean ± SD, ns. non-significant P value; * P value<0.05, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.$

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Integrin β1 blocking stimulates FCL and RA formation. (A) U2Os-AP2-GFP cells plated on FN were treated with integrin β1 blocking antibody mab13 (0.3 µg/ml) for 15 and 45 min (or vehicle for 45 min = control) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (B) Analysis of AP2 signal colocalizing with no markers (non), p-Pax, or integrin αvβ5 over time. N (images): control = 18, mab13 15 min = 13, mab13 45 min = 15, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 119) = 46.68, P < 0.0001. (C) Analysis of fraction of integrin αvβ5 colocalizing with AP2 over time from samples in A. N (images): control = 15, mab13 15 min = 11, mab13 45 min = 12, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison. F (2, 35) = 45.81, P < 0.0001. (D) U2Os-AP2-GFP cells plated on FN were treated with mab13 (0.3 µg/ml) and imaged using TIRF microscopy. 5-min time-lapses with 1-s intervals starting at 0 min (no mab13) and every 5 min after mab13 addition, until 35 min, were acquired. N (videos): 0 min = 11, 5 min = 11, 10 min = 9, 15 min = 8, 20 min = 8, 25 min = 6, 30 min = 7, 35 min = 6. Videos were acquired from two independent experiments. Similar results were observed in five individual experiments. One-way ANOVA with Tukey’s multiple comparison. F (7, 29) = 8.893, P < 0.0001. (E) A schematic illustration of results shown in this figure. Data are the mean ± SD, ns. non-significant P value; * P value<0.05, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Techniques: Blocking Assay, Control, Staining, Comparison, Microscopy

Depletion of integrin β1 or Integrin α5 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax and imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (B) Analysis of FCL proportions from samples in A. N (videos): shScr = 12, shITGB1 #1 = 11, shITGB1 #2 = 10, from three individual experiments. Tukey’s multiple comparison, F (2, 30) = 27.81, P < 0.001. (C) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB #1, #2), or integrin α5 with two different shRNAs (shITGA5 #1, #2), or control shRNA, were plated on FN-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from integrin β1 silenced samples in C. N (images): control shRNA = 30, shITGB1 #1 = 27, shITGB1 #2 = 18, from two independent experiments; similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 72) = 276.2, P < 0.0001. (E) Analysis of RA coverage from integrin α5 silenced samples in E. N (images): control shRNA = 33, shITGA5 #1 = 40, shITGA5 #2 = 37, from three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 107) = 44.46, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P-value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Depletion of integrin β1 or Integrin α5 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax and imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (B) Analysis of FCL proportions from samples in A. N (videos): shScr = 12, shITGB1 #1 = 11, shITGB1 #2 = 10, from three individual experiments. Tukey’s multiple comparison, F (2, 30) = 27.81, P < 0.001. (C) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB #1, #2), or integrin α5 with two different shRNAs (shITGA5 #1, #2), or control shRNA, were plated on FN-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from integrin β1 silenced samples in C. N (images): control shRNA = 30, shITGB1 #1 = 27, shITGB1 #2 = 18, from two independent experiments; similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 72) = 276.2, P < 0.0001. (E) Analysis of RA coverage from integrin α5 silenced samples in E. N (images): control shRNA = 33, shITGA5 #1 = 40, shITGA5 #2 = 37, from three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 107) = 44.46, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P-value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Techniques: Control, shRNA, Staining, Microscopy, Comparison

Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Silencing of integrin α5β1 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (B) Analysis of 12G10 fluorescent intensity. N = 7 from one representative experiment. Similar results were observed from two individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 13) = 149.9, P < 0.0001. (C) Western blots showing integrin β1 silencing efficiency and the effect on integrin β5 protein levels. U2Os cells silenced for integrin β1 shRNAs (shITGB1 #1, #2) or control shRNA were blotted for integrin β1, integrin α5, and α-tubulin. Representative blots out of two individual experiments. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for integrin α5 with two different shRNAs (shITGA5 #1, #2) or control shRNA were plated on FN-coated dishes and stained for integrin α5 (SNAKA51 antibody). Representative TIRF images. (E) Analysis of SNAKA51 fluorescent intensity from widefield microscopic images. N (images): shScr = 24, shITGA5 #1 = 20, shITGA5 #2 = 19 from one representative experiment. Similar results were observed in two individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 60) = 1, P < 0.0001. Data are the mean ± SD, *** P < 0.001. Scale bars, 10 µm. Source data are available for this figure: .

Techniques: Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight

Active integrin α5β1 at fibrillar adhesions inhibit FCL and RA formation. (A) U2Os-AP2-halo cells were plated to FN-coated or non-coated dishes and stained for Tensin1 and active integrin β1 12G10. Representative TIRF images. (B) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (C) Analysis of RA coverage from samples in B. N (images): shScr control = 35, shTNS1 #1/#2 = 44, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 120) = 56.26, P < 0.0001. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (E) Analysis of 12G10 fluorescent intensity from samples in D. N (images): shScr = 20, shTNS1 #1 = 15, shTNS1 #2 = 11, from one representative image. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 43) = 87.81, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Active integrin α5β1 at fibrillar adhesions inhibit FCL and RA formation. (A) U2Os-AP2-halo cells were plated to FN-coated or non-coated dishes and stained for Tensin1 and active integrin β1 12G10. Representative TIRF images. (B) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (C) Analysis of RA coverage from samples in B. N (images): shScr control = 35, shTNS1 #1/#2 = 44, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 120) = 56.26, P < 0.0001. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (E) Analysis of 12G10 fluorescent intensity from samples in D. N (images): shScr = 20, shTNS1 #1 = 15, shTNS1 #2 = 11, from one representative image. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F (2, 43) = 87.81, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.

Techniques: Staining, Control, shRNA, Comparison

Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Knockdown of focal or fibrillar adhesion components or lateral confinement promote FCL and RA formation. (A) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for Tensin1. Representative TIRF images. (B) Analysis of Tensin1 fluorescent intensity from samples in A. N (images): shScr control = 22, shTNS1 #1 = 29, shTNS1 #2 = 31, from one representative experiment. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison F (2, 79) = 38.09, P < 0.0001. (C) Representative Western blots of Tensin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were blotted for Tensin1 and α-tubulin. The arrow marks the correct Tensin1 band. The position of molecular weight markers (in kDa) are shown on the left. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for Talin1. Representative TIRF images. (E) Analysis of Talin1 fluorescent intensity from samples in D. N (images): shScr = 32, shTLN1 = 31, from two individual experiments. Two-tailed Student’s t test, P < 0.0001. (F) Representative Western blots of Talin1 silencing efficiency. U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 shRNA or control shRNA were blotted for Talin1 and GAPDH. The position of molecular weight markers (in kDa) are shown on the left. (G) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (H) Analysis of RA coverage from samples in G. N (images): n = 23. Two-tailed Student’s t-test, P < 0.0001. (I) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Talin1 with shRNA or control shRNA were plated on FN-coated dishes and stained for integrin β1 12G10. Representative TIRF images. (J) Analysis of 12G10 fluorescent intensity with samples from I. N (images): shScr = 20, shTLN1 = 15, from one representative experiment. Similar results were observed in two individual experiments. Two-tailed Student’s t test, P < 0.0004. (K) U2Os-AP2-GFP cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (L) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. Data are the mean ± SD, ** P < 0.01, *** P < 0.001. Scale bars, 10 µm; insets, 5 µm. Source data are available for this figure: .

Techniques: Knockdown, Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight, Two Tailed Test

RA disassembly is coupled to cell migration. (A) U2Os-AP2-GFP cells were grown on FN-precoated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. See for the same staining of H-shaped patterns. (B) Analysis of RA coverage from samples in A. N (images): Arrow non-coated = 28, Arrow FN = 27, H non-coated = 25, H FN = 20 from one representative image. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (C) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. See for the same staining with H-shaped patterns. (D ) Top: U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 80 min or 4 h in fresh complete medium, and stained for p-Pax. Representative TIRF images (stitched tile of five side-by-side fields of view). The wound is exactly at the right edge of the images. Bottom: Analysis of normalized RA coverage from tiles in D. RA coverage was calculated in 11-µm-wide sliding windows from the wound edge. N (tiles): 0 min = 48, 80 min = 36, 4 h = 15. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (E) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 30 min or 80 min in fresh complete medium, and stained for FN. Representative TIRF images from the migrating front (indicated as yellow lines). (F) Analysis of FN fluorescent intensity from samples in E. N (images): 0 min = 10, 30 min = 25, 80 min = 22. One-way ANOVA with Tukey’s multiple comparison, F (2, 56) = 13.77, P < 0.0001. (G) A schematic illustration of results shown in this figure. Data are the mean ± SD, ** P value < 0.01, *** P value < 0.001. Scale bars, 10 µm, except in D (50 µm) and inset (10 µm).

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: RA disassembly is coupled to cell migration. (A) U2Os-AP2-GFP cells were grown on FN-precoated or non-coated micropatterns (1,100 mm 2 ) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. See for the same staining of H-shaped patterns. (B) Analysis of RA coverage from samples in A. N (images): Arrow non-coated = 28, Arrow FN = 27, H non-coated = 25, H FN = 20 from one representative image. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (C) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm 2 ) and stained for active integrin β1 12G10. Representative TIRF images. See for the same staining with H-shaped patterns. (D ) Top: U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 80 min or 4 h in fresh complete medium, and stained for p-Pax. Representative TIRF images (stitched tile of five side-by-side fields of view). The wound is exactly at the right edge of the images. Bottom: Analysis of normalized RA coverage from tiles in D. RA coverage was calculated in 11-µm-wide sliding windows from the wound edge. N (tiles): 0 min = 48, 80 min = 36, 4 h = 15. One-way ANOVA with Tukey’s multiple comparison, F (2, 79) = 38.09, P < 0.0001. (E) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 30 min or 80 min in fresh complete medium, and stained for FN. Representative TIRF images from the migrating front (indicated as yellow lines). (F) Analysis of FN fluorescent intensity from samples in E. N (images): 0 min = 10, 30 min = 25, 80 min = 22. One-way ANOVA with Tukey’s multiple comparison, F (2, 56) = 13.77, P < 0.0001. (G) A schematic illustration of results shown in this figure. Data are the mean ± SD, ** P value < 0.01, *** P value < 0.001. Scale bars, 10 µm, except in D (50 µm) and inset (10 µm).

Techniques: Migration, Staining, Comparison

Illustration of the RA coverage calculation method. To calculate RA coverage, images of the integrin (αvβ5) and paxillin (p-Pax) channels are segmented using the Robust Automatic Threshold Selection method in ImageJ. This method generates binary masks for each channel. The paxillin channel is then subtracted from the integrin channel and results in a mask containing only the RAs. RA coverage is calculated by dividing the area covered by RAs in pixels (integrated density/255) by the area covered by the cell (or cells) in an image.

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Figure Lengend Snippet: Illustration of the RA coverage calculation method. To calculate RA coverage, images of the integrin (αvβ5) and paxillin (p-Pax) channels are segmented using the Robust Automatic Threshold Selection method in ImageJ. This method generates binary masks for each channel. The paxillin channel is then subtracted from the integrin channel and results in a mask containing only the RAs. RA coverage is calculated by dividing the area covered by RAs in pixels (integrated density/255) by the area covered by the cell (or cells) in an image.

Techniques: Selection

Journal: The Journal of Cell Biology

Article Title: Reticular adhesions are assembled at flat clathrin lattices and opposed by active integrin α5β1

doi: 10.1083/jcb.202303107

Techniques: Control, shRNA, Staining, Comparison, Western Blot, Molecular Weight

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