Journal: eLife

Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

doi: 10.7554/eLife.70278

Figure Lengend Snippet: ( A ) Representative traces of spontaneous transient outward currents (STOCs) in cerebral artery smooth muscle cells (SMCs) from control and Stim1- smKO mice, recorded by perforated patch-clamp electrophysiology over a range of membrane potentials (−60 to 0 mV). ( B, C ) Summary data showing STOC frequency ( B ) and amplitude ( C ) (control, n = 13 cells from four animals; Stim1- smKO , n = 17 cells from five mice; *p<0.05, two-way ANOVA). ( D ) Representative traces of paxilline (1 μM)-sensitive BK currents in cerebral artery SMCs from control and Stim1- smKO mice, recorded by patch-clamping in conventional whole-cell mode during a series of command voltage steps (−100 to +100 mV). ( E ) Summary data for whole-cell BK currents (control, n = 6 cells from three mice; Stim1- smKO, n = 7 cells from three mice; two-way ANOVA). ( F ) Representative traces of TRPM4 currents in cerebral artery SMCs from control and Stim1- smKO mice voltage-clamped at –70 mV, recorded using perforated patch-clamp electrophysiology. TRPM4 currents were evoked as transient inward cation currents (TICCs) by application of negative pressure (–20 mmHg) through the patch pipette and were blocked by bath application of 9-phenanthrol (9-phen; 30 μM). ( G ) Summary data showing TICC activity as TRPM4 channel open probability ( NP o ) and ( H ) TICC amplitude in control (n = 12 cells from five mice) and Stim1- smKO (n = 15 cells from five mice) mice (*p<0.05, unpaired t -test). ( I ) Representative conventional whole-cell patch-clamp recordings of 9-phenanthrol–sensitive TRPM4 currents in cerebral artery SMCs from control and Stim1- smKO mice. Currents were activated by free Ca 2+ (200 µM), included in the patch pipette solution, and were recorded using a ramp protocol from –100 to 100 mV from a holding potential of –60 mV. ( J ) Summary of whole-cell TRPM4 current density at +100 mV (control, n = 5 cells from three mice; Stim1- smKO, n = 5 cells from three mice, unpaired t -test). ns, not significant. Figure 7—source data 1. Individual data points and analysis summaries for datasets shown in .

Article Snippet: Antibody , Anti-TRPM4 (goat polyclonal) , https://Antibodies-online.com , Cat# ABIN572220; RRID: AB_10787216 , (1:400).

Techniques: Patch Clamp, Transferring, Activity Assay

Journal: eLife

Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

doi: 10.7554/eLife.70278

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-TRPM4 (goat polyclonal) , https://Antibodies-online.com , Cat# ABIN572220; RRID: AB_10787216 , (1:400).

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

Journal: Cell Reports Medicine

Article Title: TwinF interface inhibitor FP802 stops loss of motor neurons and mitigates disease progression in a mouse model of ALS

doi: 10.1016/j.xcrm.2024.101413

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-TRPM4 , Millipore , Cat#ABN418; RRID: AB_2891307.

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Magnetic Beads, Patch Clamp, Control, Transgenic Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Deletion of Trpm4 Alters the Function of the Na v 1.5 Channel in Murine Cardiac Myocytes

doi: 10.3390/ijms22073401

Figure Lengend Snippet: Co-immunoprecipitation of Na v 1.5 and TRPM4 in HEK-293 cells. ( A ) Left panel: Input signals confirming the expression of Na v 1.5 with or without FLAG tag and TRPM4. The alpha subunit of the Na/K pump was used as a loading control. Right panel: In the immunoprecipitated (IP) fraction, the signal was observed only in the presence of FLAG-Na v 1.5 and TRPM4. ( B ) Left panel: Input signals confirming the expression of Na v 1.5 with or without FLAG tag and TRPM5. Right panel: In the immunoprecipitated fraction, no signal was observed for TRPM5, even though the IP of FLAG-Na v 1.5 was successful.

Article Snippet: Proteins were visualized by anti-Na v 1.5 or anti-TRPM4 (both Pineda Antibody Service, Berlin, Germany), anti-TRPM5 (Abcam, ab154788), or anti-Na/K pump (Abcam, ab7671).

Techniques: Immunoprecipitation, Expressing, FLAG-tag

Journal: eLife

Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

doi: 10.7554/eLife.70278

Figure Lengend Snippet:

Article Snippet: Cells were then blocked with 50% SEABLOCK blocking buffer (Thermo Fisher Scientific) for 2 hr and incubated overnight at 4°C with primary antibody (anti-STIM1- [4916], Cell Signaling Technologies, Danvers, MA; anti-STIM1 [610954], BD Biosciences, Franklin Lakes, NJ; anti-BKα1- [APC-021], Alomone Labs, Jerusalem, Israel; anti-RyR2- [MA3-916], Thermo Fisher Scientific; anti-TRPM4- (ABIN572220); https://antibodies-online.com , Limerick, PA; anti-IP 3 R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

Journal: bioRxiv

Article Title: The Ca 2+ -activated cation channel TRPM4 is a positive regulator of pressure overload-induced cardiac hypertrophy

doi: 10.1101/2020.12.21.423727

Figure Lengend Snippet: ( A ) Representative western blots showing the change of key proteins in this signalling pathway in the cytoplasm ( left panel ) and nucleus ( right panel ). ( B ) Cytoplasmic ( left panel ) and nuclear ( right panel ) quantitative data were normalized by GAPDH and Histone H2B respectively. HDAC4 nuclear export was determined using the HDAC4 cytoplasmic/nuclear ratio. Results are presented as means ± SEM, n = 6/group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Samples were transferred to PVDF membranes (Bio-Rad), blocked with 5% bovine serum albumin (BSA) then labelled overnight with primary antibodies: anti-TRPM4 (1:200, Alomone Labs), anti-CaMKIIδ (1:1000; Santa Cruz Biotechnology), anti-total HDAC4 (1:1500; Cell Signalling), anti-p-HDAC4 (1:1500; Cell Signalling), anti-MEF2A (1:3000; Cell Signalling), anti-NFATc4 (1:1500 final dilution; Abcam), anti-total GSK3β (1:500; Cell Signalling), anti-p-GSK3β (Ser9, 1:1500; Cell Signalling), anti-GATA4 (1:1000; Santa Cruz Biotechnology).

Techniques: Western Blot

Journal: eLife

Article Title: STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells

doi: 10.7554/eLife.70278

Figure Lengend Snippet:

Article Snippet: Cells were then blocked with 50% SEABLOCK blocking buffer (Thermo Fisher Scientific) for 2 hr and incubated overnight at 4°C with primary antibody (anti-STIM1- [4916], Cell Signaling Technologies, Danvers, MA; anti-STIM1 [610954], BD Biosciences, Franklin Lakes, NJ; anti-BKα1- [APC-021], Alomone Labs, Jerusalem, Israel; anti-RyR2- [MA3-916], Thermo Fisher Scientific; anti-TRPM4- (ABIN572220); https://antibodies-online.com , Limerick, PA; anti-IP 3 R- (ab5804), Abcam, Cambridge, UK) diluted in PBS containing 20% SEABLOCK, 1% BSA, and 0.05% Triton X-100.

Techniques: Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Software

Journal: Cancers

Article Title: The Prognostic Value of the Circulating Tumor Cell-Based Four mRNA Scoring System: A New Non-Invasive Setting for the Management of Bladder Cancer

doi: 10.3390/cancers14133118

Figure Lengend Snippet: The characterization and functionality of the CTCs from BC patients. ( A ) The representative confocal microscopy image of the isolated CTCs stained with anti-human pan-CK and anti-human CD45 Abs. DAPI was used to counteract the nuclei. Magnification 60×. ( B ) The representative light microscopy image of the CTCs, stained with toluidine blue, migrated on the membrane to the bottom face. The arrow indicates the pores of the transwell. ( C ) The representative confocal image of the CTCs and T24 cells that invaded the Matrigel in the transwell invasion assay. Cells were stained with pan-CK and DAPI, Magnification 60×. ( D ) The percentage of patients who displayed CTCs with an invasive phenotype, as demonstrated by the transwell assay.

Article Snippet: Mouse anti-human pan-cytokeratin (C11) Ab (1:50, sc-8018, Santa Cruz Biotechnology, Heidelberg, Germany), anti-human CD45 Ab (1:50, #13917, Cell Signaling Technology, Danvers, MA, USA), anti-human TWIST1 Ab (1:50, sc-81417, Santa Cruz Biotechnology), anti-human EGFR (1:50, sc-373746, Santa Cruz Biotechnology), anti-human TRPM4 (1:50, HPA041169, Sigma Aldrich, St. Louis, MO, USA), and anti-human ZEB1 (1:50, sc-515797, Santa Cruz Biotechnology), followed by corresponding IgG Abs (Alexa Fluor ® 594, 1:100, Abcam, Cambridge, UK) were used to stain the CTCs.

Techniques: Confocal Microscopy, Isolation, Staining, Light Microscopy, Membrane, Transwell Invasion Assay, Transwell Assay