anti trpm6 antibody  (Alomone Labs)

Journal: Scientific Reports

Article Title: Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

doi: 10.1038/s41598-018-21004-w

Figure Lengend Snippet: TRPM7/TRPM6 protein expression and TRPM7 kinase activity in splenic T cells. ( A ) Western blot analysis of immunoprecipitated TRPM7 from whole cell lysates of WT and KD splenic T cells. T cells were stimulated with PMA/ionomycin or anti-CD3/CD28 antibody coated beads for 48 hrs. Mouse embryonic fibroblasts were used as a positive control. Equal amounts of protein before immunoprecipitation were ensured by probing for actin. ( B ) Incorporation of 32 P into exogenous myelin basic protein (MBP) by TRPM7 immunoprecipitated from WT and KD resting T cells. Equal quantities of MBP were verified by coomassie blue staining. ( C ) Control experiment showing that anti-TRPM6 antibody was able to recognize TRPM6, by immunoprecipitation using anti-TRPM6 antibody in GFP-TRPM6 transfected HEK cells ( D .

Article Snippet: Extracts of T cells, mouse embryonic fibroblasts (MEF) or HEK293 cells (untransfected or heterologously expressing GFP tagged human TRPM6 in pEGFP-C1 vector) were obtained as described above for the TRPM7 kinase assay and incubated with anti-TRPM7 antibody (1:500) or anti-TRPM6 antibody (1:500, ACC-046; Alomone labs, Israel) overnight at 4 °C, followed by incubation with protein A sepharose beads for 1 hr.

Techniques: Expressing, Activity Assay, Western Blot, Immunoprecipitation, Positive Control, Staining, Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

doi: 10.1152/ajprenal.00057.2015

Figure Lengend Snippet: Immunofluorescence staining of CBD-28k ( A ), TRPV5 ( B ), and TRPM6 ( C ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment.

Article Snippet: The membrane was incubated with an anti-TRPV5 antibody (1:1,000, Abcam), anti-CBD-28k antibody (1:20,000, Swant, Marly, Switzerland), anti-TRPM6 antibody (1:1,000, Alomone Laboratory, Jerusalem, Israel), and anti-CLDN16 antibody (1:3,000, Abcam) after blocking with 10% nonfat milk.

Techniques: Immunofluorescence, Staining, Mouse Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

doi: 10.1152/ajprenal.00057.2015

Figure Lengend Snippet: Immunoblotting study of CBD-28k ( A ) TRPV5 ( B ), TRPM6 ( C ), and claudin-16 ( D ) in renal tissue of wild-type and CBD-28k KO mice following CTZ and FSM treatment. The whole blots are shown with molecular size markers labeled on the left . The specific bands

Article Snippet: The membrane was incubated with an anti-TRPV5 antibody (1:1,000, Abcam), anti-CBD-28k antibody (1:20,000, Swant, Marly, Switzerland), anti-TRPM6 antibody (1:1,000, Alomone Laboratory, Jerusalem, Israel), and anti-CLDN16 antibody (1:3,000, Abcam) after blocking with 10% nonfat milk.

Techniques: Mouse Assay, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

doi: 10.3390/ijms22168744

Figure Lengend Snippet: Immunofluorescence images suggesting the presence of TRPM6 and TRPM7 proteins in pig cardiomyocytes from different cardiac chamber walls. ( A – D ) Immunofluorescence of TRPM7 (left) and TRPM6 (right) in the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV) cardiomyocytes when using Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( E ) Example of a negative control, where the primary antibody for TRPM6 and/or TRPM7 is not added, but the cardiomyocyte was subjected to Hoechst 33342 and Alexa Fluor 546. Under such conditions, only immunofluorescence of the nuclei (stained in blue) and F actin cytoskeleton (stained in red) is detected. Note: same cardiomyocyte in the left and right (merged image) panels ( F ) Quantification of the staining intensity of the immunodetected fluorescence of TRPM7 and TRPM6 in the four cardiac chamber walls: LA, RA, LV, and RV. The mean data is provided in arbitrary units (a.u.) (see Table 1 ). A blinded study design (with the origin or treatment of cells unknown to the investigator) was used for the detection of immunofluorescence during the various experimental conditions.

Article Snippet: The cells were permeabilized and incubated with primary rabbit polyclonal anti-TRPM7 (#ACC-047; Alomone Labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM6 antibody (#ACC-046; Alomone Labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Negative Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current

doi: 10.3390/ijms22168744

Figure Lengend Snippet: Comparison of the expression of TRPM6 and TRPM7 in left ventricular cardiomyocytes incubated for 2 h vs. 12 h in extracellular solutions with ( A , B ) and without ( C , D ) divalent cations (DV and DVF, respectively). ( A , C ) The cardiomyocytes were fixed after 2 h (filled columns) or 12 h (unfilled columns) of cell isolation: Alexa Fluor 488 for the TRPM7 and TRPM6 proteins (stained in green), Alexa Fluor 546 for the F-actin cytoskeleton (stained in red), and Hoechst 33342 for the nuclei (stained in blue). Scale bars indicate 20 µm. ( B , D ) Quantification of the intensity of the fluorescence expressed in arbitrary units (a.u.). # p

Article Snippet: The cells were permeabilized and incubated with primary rabbit polyclonal anti-TRPM7 (#ACC-047; Alomone Labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM6 antibody (#ACC-046; Alomone Labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C.

Techniques: Expressing, Incubation, Cell Isolation, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Effect on incubating human cardiomyocytes in acidic solution on the immunofluorescence of TRPM6 and TPRM7. ( a, b ) Quantification of the intensity of fluorescence at pH = 7.4 ( open symbols ) and at pH = 5.0 ( filled symbols ) expressed in arbitrary units (a.u.) with and without divalent cations, respectively. * P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Fluorescence

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Immunofluorescence images depicting co-expression of TRPM6 and TRPM7 proteins in human cardiomyocytes. ( a ) The immunofluorescence of TRPM7 ( green ) and TRPM6 ( red ) in the same LA, RA, LV, and RV cardiomyocyte when using conjugated antibodies (the arrowheads indicate the localization of TRPM6 protein in perinuclear area). ( b ) Quantification of the staining intensity of the immunodetected conjugated antibodies ( spotty ) and non-conjugated antibodies ( smooth ) for both proteins in cardiomyocytes from the four chambers of the heart as indicated. * P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Expressing, Staining

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Representative histotopograms of TRPM6 images in the four chamber walls of the human heart. ( a ) Control subject after traffic accident. ( b ) IHD patient. The intensity of the brownish pigments shows areas with expression of TRPM6 in the heart (× 40 magnification). ( c ) Negative control ( left ) in the LV, and positive control ( right ) of TRPM6 in tumour of the colon (× 40 magnification). Scale bars indicate 100 µm. Other notations are the same as in Fig. 7 .

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Expressing, Negative Control, Positive Control

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Effect of 2-APB and CAR on the immunofluorescence of TRPM6 and TPRM7 in human cardiomyocytes. ( a – f ): Cardiomyocyte staining with anti-TRPM7 ( a , c , e ) or anti-TRPM6 ( b , d , f ) in the absence ( a , b ) of drugs and in the presence of either 2-APB ( c , d ) or CAR ( e , f ). ( g – j ): Quantification of the intensity of fluorescence without drugs ( open ) and with the drugs ( filled ) expressed in arbitrary units (a.u.). Note lack of influence of the solvent, DMSO, at 500 μmol/L (triangles) but opposite change with 2-APB and CAR on TRPM7 vs. TRPM6, i.e. decrease of the immunofluorescence level of TRPM7 but increase of the TRPM6 fluorescence level. * P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Comparison of the levels of TRPM6 and TRPM7 in IHD vs. non-IHD. ( a–d , e–h ) Quantification of the intensity of fluorescence in cardiomyocytes obtained from patients with IHD ( filled symbols ) and without such diagnosis ( unfilled symbols ) expressed in arbitrary units (a.u.) in presence/absence of divalent cations, 2 h and 12 h, respectively. In all cells used P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: Immunofluorescence of TRPM7 and TRPM6 proteins in all cells used. Image acquisition performed using confocal laser scanning microscope ( a , atria; b , ventricle). Immunofluorescence of confocal z-stack of cardiomyocytes with immunodetected TRPM7 and TRPM6 proteins, respectively. Alexa Fluor 488 and Alexa Fluor 546 for the TRPM7 and TRPM6 protein appear in green and red, respectively. Alexa Fluor 405 for F-actin cytoskeleton appears in surrogate grey. Hoechst 33342 for nuclei appears in blue (the arrowheads indicate the localization of TRPM6 protein in the perinuclear area). ( c , d ) Quantification of immunofluorescence levels of the TRPM7 ( green ) and TRPM6 ( red ) proteins in cardiomyocytes from four chambers of the heart (left atrium, LA; right atrium, RA; left ventricle, LV; and right ventricle, RV), under experimental conditions with ( c ) and without ( d ) divalent cations in the extracellular milieu, respectively. Cardiomyocytes were fixed following 2 h ( filled columns ) or 12 h ( unfilled columns ) after cell isolation. Mean data provided in arbitrary units (a.u.) (Supplementary Table 1 online). A blinded study-design (with the investigator reading the fluorescence not knowing the cell incubation conditions) was used for the detection of protein concentration during various experimental conditions. * P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Immunofluorescence, Laser-Scanning Microscopy, Cell Isolation, Fluorescence, Incubation, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

doi: 10.1038/s41598-021-94856-4

Figure Lengend Snippet: TRPM7 and TRPM6 protein levels and RT-qPCR mRNA relative expression levels in human heart tissue homogenates. ( a, b ) TRPM7 and TRPM6 proteins are increased in the walls of all heart chambers with IHD ( filled columns ) vs. non-IHD ( unfilled columns ). A blinded study-design (with the diagnosis unknown to the investigator) was used for the detection of protein concentration in the various samples. Values (mean ± SEM) are in pg/mL and from 3–33 heart tissue homogenates. * P

Article Snippet: Non-specific binding of antibody was prevented by using blocking buffer containing 10% bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 1 h. Cells were incubated with primary rabbit polyclonal anti-TRPM6 antibody (ACC-046, Alomone labs, Jerusalem, Israel) or rabbit polyclonal anti-TRPM7 antibody (ACC-047, Alomone labs, Jerusalem, Israel) diluted (1:200) in PBS containing 3% BSA in blocking buffer overnight at 4 °C, and washed thereafter with PBS.

Techniques: Quantitative RT-PCR, Expressing, Protein Concentration