Incubation:Article Title: In vivo dual RNA-Seq uncovers key effectors of epithelial barrier disruption by an extracellular pathogen
Article Snippet: 10 μg of total proteins were loaded on a 12% SDS-PAGE (Mini-PROTEAN TGX Stain-Free precast gels, Biorad) and transferred onto nitrocellulose membrane. .. The membranes were blocked with PBS Tween20 0.1% containing 5% BSA for 1 h at RT and incubated overnight at 4 °C with the antibodies anti-ZO1 (13663; Cell Signaling), anti-ZO2 (2847; Cell Signaling), anti-Profilin-1 (3246; Cell Signaling), anti-WAVE-2 (3659; Cell Signaling), anti-pRac1 (2461; Cell Signaling), anti-pCofilin (3313; Cell Signaling), anti-Cofilin (5175; Cell Signaling), anti-GAPDH (5174; Cell Signaling), anti-MLC2 (8505; Cell Signaling), anti-pMLC2 (3671; Cell Signaling), anti-PERK (3192; Cell Signaling), anti-pPERK (PA5-40294; ThermoFisher), anti-IRE1 (3294; Cell Signaling), anti-pIRE1 (PA1-16927; ThermoFisher) and anti-3xFLAG (87537; Cell Signaling) at a 1:1000 dilution. .. Membranes were washed in PBS-0.1% Tween20 and incubated at RT with secondary HRP-conjugated antibody (7074S; Cell Signaling) at a 1:1000 dilution for 1 h. Detection was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher).
Article Title: In vivo dual RNA-Seq uncovers key effectors of epithelial barrier disruption by an extracellular pathogen.
Article Snippet: 10 μg of total proteins were loaded on a 12% SDS-PAGE (Mini-PROTEAN TGX Stain-Free precast gels, Biorad) and transferred onto nitrocellulose membrane. .. The membranes were blocked with PBS Tween20 0.1% containing 5% BSA for 1 h at RT and incubated overnight at 4 °C with the antibodies anti-ZO1 (13663; Cell Signaling), anti-ZO2 (2847; Cell Signaling), anti-Profilin-1 (3246; Cell Signaling), anti-WAVE-2 (3659; Cell Signaling), anti-pRac1 (2461; Cell Signaling), anti-pCofilin (3313; Cell Signaling), anti-Cofilin (5175; Cell Signaling), anti-GAPDH (5174; Cell Signaling), anti-MLC2 (8505; Cell Signaling), anti-pMLC2 (3671; Cell Signaling), anti-PERK (3192; Cell Signaling), anti-pPERK (PA5-40294; ThermoFisher), anti-IRE1 (3294; Cell Signaling), anti-pIRE1 (PA1-16927; ThermoFisher) and anti-3xFLAG (87537; Cell Signaling) at a 1:1000 dilution.Membranes werewashed in PBS-0.1% Tween20 and incubated at RT with secondary HRPconjugated antibody (7074S; Cell Signaling) at a 1:1000 dilution for 1 h. Detection was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). ..
Variant Assay:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. Gene name Antibody Dilution DIAPH1 BD Biosciences, mouse anti-mDia1, BD 610848 1:1000 ARP3 Santa Cruz, mouse anti-ARP3, (A-1): sc-48344; Note: recognizes both variants 1 and 2 1:500 ARP3 Santa Cruz, mouse anti-ARP3, (B-1): sc-374200; Note: epitope targets variant 1 according to the manufacturer 1:500 CFL1 Cell Signaling Technology, rabbit anti-Cofilin-1, D3F9 XP #5175 1:2000 ACTN1 Abcam, rabbit anti-alpha Actinin, ab175944 1:1000 PTK2 Santa Cruz, mouse anti-FAK, (D-1): sc-271126 1:1000 FMNL1 Santa Cruz, mouse anti-FMNL1, (C-5): sc-390023 1:1000 FMNL3 Abcam, mouse anti-FMNL2, ab57963; Note: also recognizes FMNL3 1 1:1000 DIAPH3 ProteinTech, rabbit anti-DIAPH3, 14342-1-AP 1:1000 INF2 Bethyl Laboratories, rabbit anti-INF2, A303-428A 1:1000 FHOD1 Santa Cruz, mouse anti-FHOD1, (D-6): sc-365437 1:1000 VASP BD Biosciences, mouse anti-VASP, BD 610447 1:1000 ENAH Santa Cruz, mouse anti-Mena, (21): sc-135988 1:500 CTTN Abcam, rabbit anti-Cortactin, [EP1922Y]: ab81208 1:1000 CAPZB Santa Cruz, mouse anti-CapZ-β, (52): sc-136502 1:1000 WASF2 Cell Signaling Technology, rabbit anti-WAVE-2, (D2C8) XP® #3659 1:1000 WASL Cell Signaling Technology, rabbit anti-N-WASP (30D10) #4848 1:1000 VCL Santa Cruz, mouse anti-vinculin, (7F9): sc-73614 1:2000 VCL Sigma-Aldrich, mouse anti-vinculin, V9264 1:500 (PLA) FERMT2 Millipore, mouse anti-Kindlin-2, clone 3A3: MAB2617 1:1000 ARPC2 Millipore, rabbit anti-p34-Arc/ARPC2, 07-227-I-100UG 1:1000 1:250 (PLA) ..
Proximity Ligation Assay:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. Gene name Antibody Dilution DIAPH1 BD Biosciences, mouse anti-mDia1, BD 610848 1:1000 ARP3 Santa Cruz, mouse anti-ARP3, (A-1): sc-48344; Note: recognizes both variants 1 and 2 1:500 ARP3 Santa Cruz, mouse anti-ARP3, (B-1): sc-374200; Note: epitope targets variant 1 according to the manufacturer 1:500 CFL1 Cell Signaling Technology, rabbit anti-Cofilin-1, D3F9 XP #5175 1:2000 ACTN1 Abcam, rabbit anti-alpha Actinin, ab175944 1:1000 PTK2 Santa Cruz, mouse anti-FAK, (D-1): sc-271126 1:1000 FMNL1 Santa Cruz, mouse anti-FMNL1, (C-5): sc-390023 1:1000 FMNL3 Abcam, mouse anti-FMNL2, ab57963; Note: also recognizes FMNL3 1 1:1000 DIAPH3 ProteinTech, rabbit anti-DIAPH3, 14342-1-AP 1:1000 INF2 Bethyl Laboratories, rabbit anti-INF2, A303-428A 1:1000 FHOD1 Santa Cruz, mouse anti-FHOD1, (D-6): sc-365437 1:1000 VASP BD Biosciences, mouse anti-VASP, BD 610447 1:1000 ENAH Santa Cruz, mouse anti-Mena, (21): sc-135988 1:500 CTTN Abcam, rabbit anti-Cortactin, [EP1922Y]: ab81208 1:1000 CAPZB Santa Cruz, mouse anti-CapZ-β, (52): sc-136502 1:1000 WASF2 Cell Signaling Technology, rabbit anti-WAVE-2, (D2C8) XP® #3659 1:1000 WASL Cell Signaling Technology, rabbit anti-N-WASP (30D10) #4848 1:1000 VCL Santa Cruz, mouse anti-vinculin, (7F9): sc-73614 1:2000 VCL Sigma-Aldrich, mouse anti-vinculin, V9264 1:500 (PLA) FERMT2 Millipore, mouse anti-Kindlin-2, clone 3A3: MAB2617 1:1000 ARPC2 Millipore, rabbit anti-p34-Arc/ARPC2, 07-227-I-100UG 1:1000 1:250 (PLA) ..
Western Blot:Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles.
Article Snippet: Cells were selected in a culture medium containing hygromycin B (InvivoGen, ant-hg-1) for the stable transfectants at appropriate concentrations. .. The following antibodies were used for western blotting (WB) and/or immunofluorescence staining (IF) at the following dilutions: mouse anti-CIP4 (BD, clone 21, 612556, 1:100 for IF, 1:1000 for WB), mouse anti-GAPDH (Santa Cruz, sc-166574, 1:2000 forWB), rabbit anti-WAVE2 (Cell signaling, clone D2C8, #3659, 1:100 for IF, 1:1000 for WB), rabbit anti-N-WASP (Cell signaling, clone 30D10, #4848, 1:1000 for WB), mouse anti-dynamin II (BD, #610263, 1:1000 for WB), mouse antiendophilin II (Santa Cruz, #sc-365704, 1:1000 for WB), rabbit anticlathrin light chain A (CLTA) (proteintech, #10852-1-AP, 1:1000 for WB), mouse anti-Rac1 (Millipore, 05-389, 1:4000 for WB), mouse antiRac1 (BD, #610651, 1:200 for WB of EV uptake, 1:400 for ELISA), rabbit anti-Cdc42 (Cell signaling, #2462, 1:1000 for WB), rabbit anti-HaloTag (Promega, #G9281, 1:1000 for WB, 1:1000 for IF, 1:1000 for ELISA), mouse anti-HaloTag (Promega, #G9211, 1:1000 for WB, 1:1000 for ELISA), mouse monoclonal anti-MIM (Abnova, #H00009788-M01, 1:1000 for WB), mouse anti-CD81 (Santa Cruz, #sc-166029, 1:2000 for WB), mouse anti-Annexin I (Santa Cruz, sc-12740, 1:1000 for WB), rabbit anti-EEA1 (Cell signaling, #3288, 1:100 for IF), mouse anti-Lamp1 (SantaCruz, #sc-20011,1:50 for IF),mouse anti-β-actin (SantaCruz, #sc47776, 1:3000 for WB), mouse anti-mCherry (Novus, #NBP1-96752, 1:1000 for IF), rabbit anti-GFP (MBL Life Science, #598, 1:4000 forWB), and rabbit anti-RFP (MBL Life Science, #PM005, 1:3000 for WB). .. The secondary antibodies are as follows: Alexa Fluor-conjugated highly cross-adsorbed goat anti-mouse or anti-rabbit IgG antibodies (Invitrogen, #A11029, #A11031, #A11034 or #A11011, 1:500 for IF), Alexa Nature Communications | (2025) 16:10900 13 Fluor 488 Phalloidin (Invitrogen, #A12379, 1:1000 for IF), Alexa Fluor 647 Phalloidin (Invitrogen, #A22287, 1:500 for IF), alkali-phosphatase (AP)-conjugated anti-mouse IgG (Promega, #S3721, 1:10,000 for WB; Sigma, #A3562, 1:30,000 for WB) or AP-conjugated anti-rabbit IgG (Promega, #S3731, 1:10,000 for WB), Horseradish peroxidase (HRP)conjugated anti-mouse IgG (Promega, #W4021, 1:30,000 for WB) or HRP-conjugated anti-rabbit IgG (Promega, #W4011, 1:30,000 for WB).
Immunofluorescence:Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles.
Article Snippet: Cells were selected in a culture medium containing hygromycin B (InvivoGen, ant-hg-1) for the stable transfectants at appropriate concentrations. .. The following antibodies were used for western blotting (WB) and/or immunofluorescence staining (IF) at the following dilutions: mouse anti-CIP4 (BD, clone 21, 612556, 1:100 for IF, 1:1000 for WB), mouse anti-GAPDH (Santa Cruz, sc-166574, 1:2000 forWB), rabbit anti-WAVE2 (Cell signaling, clone D2C8, #3659, 1:100 for IF, 1:1000 for WB), rabbit anti-N-WASP (Cell signaling, clone 30D10, #4848, 1:1000 for WB), mouse anti-dynamin II (BD, #610263, 1:1000 for WB), mouse antiendophilin II (Santa Cruz, #sc-365704, 1:1000 for WB), rabbit anticlathrin light chain A (CLTA) (proteintech, #10852-1-AP, 1:1000 for WB), mouse anti-Rac1 (Millipore, 05-389, 1:4000 for WB), mouse antiRac1 (BD, #610651, 1:200 for WB of EV uptake, 1:400 for ELISA), rabbit anti-Cdc42 (Cell signaling, #2462, 1:1000 for WB), rabbit anti-HaloTag (Promega, #G9281, 1:1000 for WB, 1:1000 for IF, 1:1000 for ELISA), mouse anti-HaloTag (Promega, #G9211, 1:1000 for WB, 1:1000 for ELISA), mouse monoclonal anti-MIM (Abnova, #H00009788-M01, 1:1000 for WB), mouse anti-CD81 (Santa Cruz, #sc-166029, 1:2000 for WB), mouse anti-Annexin I (Santa Cruz, sc-12740, 1:1000 for WB), rabbit anti-EEA1 (Cell signaling, #3288, 1:100 for IF), mouse anti-Lamp1 (SantaCruz, #sc-20011,1:50 for IF),mouse anti-β-actin (SantaCruz, #sc47776, 1:3000 for WB), mouse anti-mCherry (Novus, #NBP1-96752, 1:1000 for IF), rabbit anti-GFP (MBL Life Science, #598, 1:4000 forWB), and rabbit anti-RFP (MBL Life Science, #PM005, 1:3000 for WB). .. The secondary antibodies are as follows: Alexa Fluor-conjugated highly cross-adsorbed goat anti-mouse or anti-rabbit IgG antibodies (Invitrogen, #A11029, #A11031, #A11034 or #A11011, 1:500 for IF), Alexa Nature Communications | (2025) 16:10900 13 Fluor 488 Phalloidin (Invitrogen, #A12379, 1:1000 for IF), Alexa Fluor 647 Phalloidin (Invitrogen, #A22287, 1:500 for IF), alkali-phosphatase (AP)-conjugated anti-mouse IgG (Promega, #S3721, 1:10,000 for WB; Sigma, #A3562, 1:30,000 for WB) or AP-conjugated anti-rabbit IgG (Promega, #S3731, 1:10,000 for WB), Horseradish peroxidase (HRP)conjugated anti-mouse IgG (Promega, #W4021, 1:30,000 for WB) or HRP-conjugated anti-rabbit IgG (Promega, #W4011, 1:30,000 for WB).
Staining:Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles.
Article Snippet: Cells were selected in a culture medium containing hygromycin B (InvivoGen, ant-hg-1) for the stable transfectants at appropriate concentrations. .. The following antibodies were used for western blotting (WB) and/or immunofluorescence staining (IF) at the following dilutions: mouse anti-CIP4 (BD, clone 21, 612556, 1:100 for IF, 1:1000 for WB), mouse anti-GAPDH (Santa Cruz, sc-166574, 1:2000 forWB), rabbit anti-WAVE2 (Cell signaling, clone D2C8, #3659, 1:100 for IF, 1:1000 for WB), rabbit anti-N-WASP (Cell signaling, clone 30D10, #4848, 1:1000 for WB), mouse anti-dynamin II (BD, #610263, 1:1000 for WB), mouse antiendophilin II (Santa Cruz, #sc-365704, 1:1000 for WB), rabbit anticlathrin light chain A (CLTA) (proteintech, #10852-1-AP, 1:1000 for WB), mouse anti-Rac1 (Millipore, 05-389, 1:4000 for WB), mouse antiRac1 (BD, #610651, 1:200 for WB of EV uptake, 1:400 for ELISA), rabbit anti-Cdc42 (Cell signaling, #2462, 1:1000 for WB), rabbit anti-HaloTag (Promega, #G9281, 1:1000 for WB, 1:1000 for IF, 1:1000 for ELISA), mouse anti-HaloTag (Promega, #G9211, 1:1000 for WB, 1:1000 for ELISA), mouse monoclonal anti-MIM (Abnova, #H00009788-M01, 1:1000 for WB), mouse anti-CD81 (Santa Cruz, #sc-166029, 1:2000 for WB), mouse anti-Annexin I (Santa Cruz, sc-12740, 1:1000 for WB), rabbit anti-EEA1 (Cell signaling, #3288, 1:100 for IF), mouse anti-Lamp1 (SantaCruz, #sc-20011,1:50 for IF),mouse anti-β-actin (SantaCruz, #sc47776, 1:3000 for WB), mouse anti-mCherry (Novus, #NBP1-96752, 1:1000 for IF), rabbit anti-GFP (MBL Life Science, #598, 1:4000 forWB), and rabbit anti-RFP (MBL Life Science, #PM005, 1:3000 for WB). .. The secondary antibodies are as follows: Alexa Fluor-conjugated highly cross-adsorbed goat anti-mouse or anti-rabbit IgG antibodies (Invitrogen, #A11029, #A11031, #A11034 or #A11011, 1:500 for IF), Alexa Nature Communications | (2025) 16:10900 13 Fluor 488 Phalloidin (Invitrogen, #A12379, 1:1000 for IF), Alexa Fluor 647 Phalloidin (Invitrogen, #A22287, 1:500 for IF), alkali-phosphatase (AP)-conjugated anti-mouse IgG (Promega, #S3721, 1:10,000 for WB; Sigma, #A3562, 1:30,000 for WB) or AP-conjugated anti-rabbit IgG (Promega, #S3731, 1:10,000 for WB), Horseradish peroxidase (HRP)conjugated anti-mouse IgG (Promega, #W4021, 1:30,000 for WB) or HRP-conjugated anti-rabbit IgG (Promega, #W4011, 1:30,000 for WB).
Enzyme-linked Immunosorbent Assay:Article Title: Efficient cellular transformation via protein delivery through the protrusion-derived extracellular vesicles.
Article Snippet: Cells were selected in a culture medium containing hygromycin B (InvivoGen, ant-hg-1) for the stable transfectants at appropriate concentrations. .. The following antibodies were used for western blotting (WB) and/or immunofluorescence staining (IF) at the following dilutions: mouse anti-CIP4 (BD, clone 21, 612556, 1:100 for IF, 1:1000 for WB), mouse anti-GAPDH (Santa Cruz, sc-166574, 1:2000 forWB), rabbit anti-WAVE2 (Cell signaling, clone D2C8, #3659, 1:100 for IF, 1:1000 for WB), rabbit anti-N-WASP (Cell signaling, clone 30D10, #4848, 1:1000 for WB), mouse anti-dynamin II (BD, #610263, 1:1000 for WB), mouse antiendophilin II (Santa Cruz, #sc-365704, 1:1000 for WB), rabbit anticlathrin light chain A (CLTA) (proteintech, #10852-1-AP, 1:1000 for WB), mouse anti-Rac1 (Millipore, 05-389, 1:4000 for WB), mouse antiRac1 (BD, #610651, 1:200 for WB of EV uptake, 1:400 for ELISA), rabbit anti-Cdc42 (Cell signaling, #2462, 1:1000 for WB), rabbit anti-HaloTag (Promega, #G9281, 1:1000 for WB, 1:1000 for IF, 1:1000 for ELISA), mouse anti-HaloTag (Promega, #G9211, 1:1000 for WB, 1:1000 for ELISA), mouse monoclonal anti-MIM (Abnova, #H00009788-M01, 1:1000 for WB), mouse anti-CD81 (Santa Cruz, #sc-166029, 1:2000 for WB), mouse anti-Annexin I (Santa Cruz, sc-12740, 1:1000 for WB), rabbit anti-EEA1 (Cell signaling, #3288, 1:100 for IF), mouse anti-Lamp1 (SantaCruz, #sc-20011,1:50 for IF),mouse anti-β-actin (SantaCruz, #sc47776, 1:3000 for WB), mouse anti-mCherry (Novus, #NBP1-96752, 1:1000 for IF), rabbit anti-GFP (MBL Life Science, #598, 1:4000 forWB), and rabbit anti-RFP (MBL Life Science, #PM005, 1:3000 for WB). .. The secondary antibodies are as follows: Alexa Fluor-conjugated highly cross-adsorbed goat anti-mouse or anti-rabbit IgG antibodies (Invitrogen, #A11029, #A11031, #A11034 or #A11011, 1:500 for IF), Alexa Nature Communications | (2025) 16:10900 13 Fluor 488 Phalloidin (Invitrogen, #A12379, 1:1000 for IF), Alexa Fluor 647 Phalloidin (Invitrogen, #A22287, 1:500 for IF), alkali-phosphatase (AP)-conjugated anti-mouse IgG (Promega, #S3721, 1:10,000 for WB; Sigma, #A3562, 1:30,000 for WB) or AP-conjugated anti-rabbit IgG (Promega, #S3731, 1:10,000 for WB), Horseradish peroxidase (HRP)conjugated anti-mouse IgG (Promega, #W4021, 1:30,000 for WB) or HRP-conjugated anti-rabbit IgG (Promega, #W4011, 1:30,000 for WB).
other:
Recombinant:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
Polymerase Chain Reaction:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
Purification:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
Electron Microscopy:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
Mutagenesis:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
Gel Extraction:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
In Situ:Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti-mDia1/DIAPH1 BD Biosciences Cat# BD 610848; RRID: AB_398167 mouse anti-ARP3 (A-1) Santa Cruz Cat# sc-48344; RRID: AB_626700 mouse anti-ARP3, (B-1) Santa Cruz Cat# sc-374200; RRID: AB_10990131 rabbit anti-Cofilin-1 D3F9 Cell Signaling Technology Cat# 5175; RRID: AB_10622000 rabbit anti-alpha Actinin Abcam Cat# ab175944 mouse anti-FAK (D-1) Santa Cruz Cat# sc-271126; RRID: AB_10614323 mouse anti-FMNL1, (C-5) Santa Cruz Cat# sc-390023 mouse anti-FMNL2 Abcam Cat# ab57963; RRID: AB_941625 rabbit anti-DIAPH3 ProteinTech Cat# 14342-1-AP; RRID: AB_2092930 rabbit anti-INF2 Bethyl Laboratories Cat# A303-428A; RRID: AB_10952400 mouse anti-FHOD1, (D-6) Santa Cruz Cat# sc-365437; RRID: AB_10843854 mouse anti-VASP BD Biosciences Cat# BD 610447 mouse anti-Mena (21) Santa Cruz Cat# sc-135988; RRID: AB_2098633 rabbit anti-Cortactin, [EP1922Y] Abcam Cat# ab81208; RRID: AB_1640383 mouse anti-CapZ-β, (52) Santa Cruz Cat# sc-136502; RRID: AB_10610091 rabbit anti-WAVE-2, (D2C8) XP Cell Signaling Technology Cat# 3659 rabbit anti-N-WASP (30D10) Cell Signaling Technology Cat# 4848 mouse anti-vinculin, (7F9) Santa Cruz Cat# sc-73614; RRID: AB_2941767 mouse anti-vinculin Sigma-Aldrich Cat# V9264; RRID: AB_10603627 mouse anti-Kindlin-2, clone 3A3 Millipore Cat# MAB2617; RRID: AB_10631873 rabbit anti-p34-Arc/ARPC2 Millipore Cat# 07-227 mNeonGreen-Trap Magnetic Agarose beads Chromotek Cat# ntma-20; RRID: AB_2827594 Chemicals, peptides, and recombinant proteins blasticidine S HCl Thermo R21001 Polyethylenimine Sigma 408727 Platinum SuperFi II PCR Master Mix Thermo Cat # 12368010 AF488-conjugated Phalloidin Thermo A12379 AF568-conjugated Phalloidin Thermo A12380 Bovine collagen, Type 1 Advanced Biomatrix Cat# 5133 rat-tail collagen Corning Cat# 354249 CK-666 Millipore Cat# 182515 purified Arp2/3 complex Cytoskeleton RP-01-PA QGel 920 CHT QGel 920 20% Paraformaldehyde Electron Microscopy Sciences 15713 Critical commercial assays Q5 site-directed PCR mutagenesis kit NEB E0554S DNeasy Blood and Tissue kit Qiagen Cat # 69504 QIAquick Gel Extraction Kit Qiagen Cat # 28704 Duolink® In Situ Detection Red kit Sigma DUO92008 Experimental models: Cell lines U2OS Gifted by Dick McIntosh (University of Colorado) RPE-1 ATCC CRL-4000; RRID: CVCL_4388 (Continued on next page) iScience 28, 112623, June 20, 2025 e1 .. Cell lines and culture conditions Human Osteosarcoma (U2OS; female) and human retinal pigment epithelial (RPE-1; female) cells were cultured in DMEM or RPMI media, respectively supplemented with 10% fetal bovine serum (Sigma; F0926-500ML) and maintained in a humidified incubator at 37◦C and 5% CO2.
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