Virus:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Recombinant:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
MTT Assay:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Membrane:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Planar Chromatography:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Proximity Ligation Assay:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Purification:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
Incubation:Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Polyclonal rabbit anti-bPix (Millipore 1:1000, 07-1450-L), Monoclonal mouse anti-Myosin18A (1:200, Santa Cruz, H-10, 365328), Monoclonal mouse anti-Cdc42 monoclonal antibody (1:250, Cytoskeleton, Cat. # ACD03), anti-Rac1 (1:250, Cytoskeleton, Cat. # ACC03), Rabbit polyclonal anti-Git1 (1:1000, Novus, NBP1-86144), Polyclonal rabbit anti-GAPDH (1:10,000: AB2302; EMD Millipore), Monoclonal mouse anti-phospho-myosin-light-chain (1:500, Cell Signaling #3675), myosin-light-chain (1/500, Cell Signaling #8505S).For the detection of biotin, membranes were incubated with Alexa Fluor-680 conjugated streptavidin (1.8 mg/mL, Jackson ImmunoResearch) diluted 1:2000 in 5% BSA (in TBST).
Article Title: Calcium-sensing receptor regulates Kv7 channels via G i/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.
Article Snippet: The following compounds and antibodies were used in this study: NPS-R568, a CaSR PAM agonist (Tocris Bioscience, Abingdon, UK); NPS-2143, a CaSR NAM (Tocris); retigabine, a Kv7 channel opener (Tocris); XE991, a Kv7 channel blocker (Tocris); forskolin, a transmem- brane adenylate cyclase (AC) activator (Tocris); 8-Br-cAMP, a phosphodiesterase-resistant cAMP analogue and activator of protein kinase A (PKA) (Merck Life Science, Dorset, UK); Bordetella pertussis toxin (PTX), a specific inhibitor targeting the alpha subunit of the Gi/o protein (Merck); nystatin (Merck); U73122, a phospholipase C (PLC) inhibitor (Tocris); 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N0tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), an [Ca2+]i chelator (Tocris); NiCl2 hexahydrate, a non-selective blocker of Ca 2+ influx (Fisher Scientific, Loughborough, UK); bradykinin (Merck); substance P (Tocris); adenosine 5'-triphosphate (ATP; Merck); tumour necrosis factor (TNFα) (Merck); mouse monoclonal anti-CaSR (Abcam Cat# ab19347, RRID:AB_444867, Abcam, Cambridge, UK); rabbit polyclonal anti-CaSR (Sigma-Aldrich Cat# SAB4503369, RRID: AB_10747268); rabbit polyclonal anti-transient receptor potential vanilloid 1 (Abcam Cat# ab3487, RRID:AB_2209009); rabbit poly- clonal anti-KCNQ2 (Abcam Cat# ab22897, RRID:AB_775890); rabbit polyclonal anti-KCNQ3 (Thermo Fisher Scientific Cat# PA1–930, RRID:AB_2131708, Life Technologies, Paisley, UK); mouse monoclo- nal anti-microtubule-associated protein 2 (Sigma-Aldrich Cat# M1406, RRID:AB_477171); rabbit polyclonal anti-peripherin (Abcam Cat# ab4666, RRID:AB_449340); mouse monoclonal anti-phosphoserine conjugated to Duolink® PLA MINUS probe (DUO87012, Merck); Alexa 488 donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21202, RRID: AB_141607); Alexa 594 donkey anti-mouse IgG (H + L) highly cross- adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21203, RRID:AB_2535789); Alexa 488 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792); Alexa 594 donkey anti- rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637); Alexa 488 goat anti-mouse IgG (H + L) pre-adsorbed secondary antibody (Abcam Cat# ab150117, RRID:AB_2688012).
Article Title: 3D matrix adhesion feedback controls nuclear force coupling to drive invasive cell migration.
Article Snippet: Primary antibodies: Mouse monoclonal anti-Paxillin (1:50, BD biosciences #610569), Polyclonal rabbit Anti-bPix (Millipore 1:200, 07-1450-L), Monoclonal mouse anti-Myosin18A, (1:200 SCBT 365328), Rabbit anti- VASP (9A2) (1:200 Cell Signaling Technology 3132S), Rabbit polyclonal anti-myosin Heavy Chain 9 (1:100 Cell Signaling Solution Technology #3403), Mouse monoclonal antimyosin Heavy chain 10 (3H2) (1:200 Abcam ab684), Rabbit Polyclonal anti-pericentrin (1:2000, Abcam ab4448), Mouse monoclonal YAP antibody (1:1000, Santa Cruz Biotechnology sc-101199), Rat monoclonal anti-⍺5b1-integrin (1:200, Mab11, in-house purification from hybridoma), Rabbit polyclonal anti-Git1 (1:200, Novus, NBP1-86144).
Article Title: An MITF- and mTOR-dependent FLCN pathway suppresses TFE3-driven metastasis in melanoma
Article Snippet: Antibody buffer (wash buffer with 2mM EDTA and 0.025% digitonin) containing anti-MITF (Sigma, HPA003259), anti-TFE3 (Cell Signaling Technologies (CST), 14779), Anti-H3K27Ac (CST, #8173), Anti-H3K27Me3 (CST, #9733), H3K4Me3 (CST, #9751), H3K9Ac (CST, #9649), or Rabbit IgG (Millipore, 12-370) was added and cells were incubated overnight at 4°C on nutator.
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