anti k ca 1 1 extracellular polyclonal antibody polyclonal antibody rabbit  (Alomone Labs)

Journal:

Article Title:

doi:

Figure Lengend Snippet: Comparison of K Ca 1.1 expression and activity between LNCaP cells cultured as 2D monolayers and 3D spheroids. ( A ): Phenotypic properties of LNCaP cells cultured in ultra-low attachment PrimeSurface 96U plates (upper panel: on day 0; lower panel: on day 7). Brightfield images were obtained with the Axio Observer Z1 microscope system (Carl Zeiss, Oberkochen, Germany). Bars show 500 μm. ( B ): Time course of the voltage-sensitive fluorescent dye imaging of K Ca 1.1 activator (1 μM NS1619)-induced hyperpolarizing responses in isolated cells from ‘2D’ monolayers and ‘3D’ spheroids of LNCaP. The fluorescent intensity of DiBAC 4 (3) before the application of NS1619 is expressed as 1.0. Images were measured every 5 s. ( C ): Summarized results of NS1619-induced hyperpolarizing responses in cells isolated from at least three different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescent intensity were obtained by measuring the average for 1 min (12 images). ( D ): K Ca 1.1 protein expression in the lipid-raft-enriched protein lysates of both groups. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa, upper panel) and anti-ACTB (43 kDa, lower panel) antibodies. ( E ): Summarized results were obtained as the optical density of K Ca 1.1 and ACTB band signals. After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in ‘2D’ was expressed as 1.0 ( n = 4 for each). ( F ): Real-time PCR examination of K Ca 1.1 in both groups ( n = 4 for each). Expression levels were shown as a ratio to ACTB. *: p < 0.05 vs. ‘2D’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Microscopy, Imaging, Isolation, Real-time Polymerase Chain Reaction

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effects of chemotherapy agents on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a pretreatment with a K Ca 1.1 inhibitor on chemoresistance acquired by 3D-cultured LNCaP cells. ( A – D ): Effects of the treatment with 100 nM DTX, 100 nM PTX, 1 μM DOX, and 10 μM CIS for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% dimethylsulfoxide, DMSO in DTX, PTX, and DOX and water in CIS instead of chemotherapy agents was expressed as 1.0. ( E – H ): Effects of the treatment with chemotherapy agents for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effects of antiandrogens on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a K Ca 1.1 inhibitor on the antiandrogen resistance acquired by 3D-cultured LNCaP cells. ( A , B ): Effects of the treatment with 10 μM BCT and 10 μM EZT for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% DMSO instead of antiandrogens was expressed as 1.0. ( C , D ): Effects of the treatment with antiandrogens for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Expression of MRP transcripts in both 2D- and 3D-cultured LNCaP cells and the effects of K Ca 1.1 blockade with PAX on their expression in 3D-cultured LNCaP cells. (A–D): Real-time PCR examination of MRP1 ( A ), MRP3 ( B ), MRP4 ( C ), and MRP5 ( D ) in ‘2D’ monolayers and ‘3D’ spheroids of LNCaP cells ( n = 4 for each). ( E – H ): Real-time PCR examination of MRP transcripts in vehicle- and PAX-treated, 3D-cultured LNCaP cells ( n = 4 for each). Expression levels were shown as a ratio to ACTB. **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal:

Article Title:

doi:

Figure Lengend Snippet: Regulation of K Ca 1.1 protein degradation through the FBXW7 ubiquitin E3 ligase in LNCaP cells. ( A , B ) Protein expression of FBXW7 in whole cell protein lysates of ‘2D’- and ‘3D’-cultured LNCaP cells. Blots were probed with anti-FBXW7 (approximately 70 kDa) and anti-ACTB (43 kDa) antibodies ( A ). Summarized results ( B ) were obtained as the optical densities of FBXW7 and ACTB band signals ( n = 4 for each). After compensation for the optical densities of the protein band signals with that of the ACTB signal, the optical density in ‘2D’ was expressed as 1.0 ( n = 4 for each). ( C , D ): Protein expression of K Ca 1.1 in control siRNA (si-Cont)- and FBXW7 siRNA (si-FBXW7)-transfected (for 72 h), 2D-cultured LNCaP cells. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa) and anti-ACTB (43 kDa) antibodies ( C ). Summarized results ( D ) were obtained as the optical densities of K Ca 1.1 and ACTB band signals. The optical density in ‘si-Cont’ was expressed as 1.0 ( n = 4 for each). **: p < 0.01 vs. ‘2D’; # : p < 0.05 vs. ‘si-Cont’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Cell Culture, Transfection

Journal:

Article Title:

doi:

Figure Lengend Snippet: Gene and protein expression of K Ca 1.1 in human breast cancer cell lines and effects of its pharmacological and/or siRNA-mediated blockade on the viability and K Ca 1.1 activity in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in seven human breast cancer cell lines ( n = 3 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Expression of K Ca 1.1 proteins (about 130 kDa) in MDA-MB-453, YMB-1, and MCF-7 cells. Protein lysates of the examined cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( C , D ) Effects of the treatment with the K Ca 1.1 blocker, paxilline (10 μM) for 72 h ( C ) and the transfection with K Ca 1.1 siRNA for 96 h ( D ) on the viability in MDA-MB-453 cells. Cell viability in the vehicle-treated or control siRNA-transfected group is arbitrary expressed as 1.0, and the data are shown as “relative cell viability” ( n = 5 for each); ( E ) Current-voltage relationship for the current amplitude at the end of the depolarization pulse in MDA-MB-453 cells following treatment with 1 µM paxilline (see ). Results are expressed as means ± SEM. * p < 0.05; ** p < 0.01 vs. the vehicle control or control siRNA.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Journal:

Article Title:

doi:

Figure Lengend Snippet: Down-regulation of K Ca 1.1 transcripts and proteins by the treatment with VDR agonists for 72 h in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in vehicle-, 1 μM calcitriol-, and 1 μM calcipotriol-treated MDA-MB-453 cells ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Band patterns on agarose gels for the PCR products of K Ca 1.1 exons (exon 1–4, 5–14, 15–23, and 24–30) in vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells. A DNA molecular weight marker is indicated on the right of the gel; ( C ) Protein lysates of vehicle-, 1 µM calcitriol-, and 1 µM calcipotriol-treated MDA-MB-453 cells were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( D ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in C . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( E ) Effects of the treatment with 1 µM calcitriol or 1 µM calcipotriol on the cell surface expression of K Ca 1.1 proteins by a flow cytometric analysis. Non-permeabilized MDA-MB-453 cells were stained with an Alexa Fluor @ 488-conjugated anti-K Ca 1.1 antibody (extracellular). Data were expressed as the relative cell population of K Ca 1.1-positive cells to those in the vehicle control (1.0) ( n = 4 for each). Results are expressed as means ± SEM. ** p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Molecular Weight, Marker, Western Blot, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Inhibitory effects of K Ca 1.1 activities (1 µM paxilline-induced depolarization responses) in MDA-MB-453 cells treated with VDR agonists for 72 h and effects of co-treatment with calcitriol (1 µM) and 1 μM paxilline on the viability of MDA-MB-453 cells. ( A ) Measurement of paxilline-induced depolarization responses in vehicle (black symbol)-, calcitriol (red symbol)-, and calcipotriol (blue symbol)-treated MDA-MB-453 cells. The fluorescence intensity of DiBAC 4 (3) before the application of paxilline at 0 s is expressed as 1.0. The time courses of changes in the relative fluorescence intensity of DiBAC 4 (3) are shown; ( B ) Summarized data are shown as the paxilline-induced ∆ relative fluorescence intensity of DiBAC 4 (3) in vehicle-, calcitriol-, and calcipotriol-treated MDA-MB-453 cells. Cells were obtained from four different batches. Numbers used for the experiments are shown in parentheses; ( C ) Effects of the treatment with calcitriol (1 µM) alone, paxilline (10 µM) alone, and calcitriol plus paxilline for 72 h on the viability in MDA-MB-453 cells. Cell viability in the vehicle-treated is arbitrary expressed as 1.0, and the data are shown as “relative cell viability” ( n = 5 for each). Results are expressed as means ± SEM. **: p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Fluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effects of the proteasome inhibitor, MG132 (100 nM) on VDR agonist-induced K Ca 1.1 protein degradation and K Ca 1.1 activity in MDA-MB-453 cells. ( A ) Protein lysates from MDA-MB-453 cells after drug treatments were probed by immunoblotting with anti-K Ca 1.1 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( B ) Summarized results are obtained as the optical density of K Ca 1.1 and ACTB band signals in A . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in the vehicle control was expressed as 1.0 (dotted line, n = 3 for each); ( C ) Summarized data are shown as the paxilline-induced ∆ relative fluorescence intensity of DiBAC 4 (3) in vehicle-, calcitriol-, and calcipotriol-treated MDA-MB-453 cells. Cells were obtained from three different batches. Numbers used for the experiments are shown in parentheses. Results are expressed as means ± SEM.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Activity Assay, Western Blot, Fluorescence

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effects of the pharmacological and siRNA-mediated blockade of HDACs on expression levels of K Ca 1.1 transcripts in MDA-MB-453 cells. ( A ) Real-time PCR assay for K Ca 1.1 in MDA-MB-453 cells treated with the following HDAC inhibitors for 48 h ( n = 4 for each): vorinostat (suberanilohydroxamic acid), a pan-HDAC inhibitor; AATB (4-(acetylamino)- N -[2-amino-5-(2-thienyl)phenyl]-benzamide), a HDAC1 (30 nM) and HDAC2 (300 nM) inhibitor; T247 (N-(2-aminophenyl)-4-[1-(2-thiophen-3-ylethyl)-1H-[1], [2], [3]triazol-4-yl]benzamide), a selective HDAC3 inhibitor; and NCT-14b (( S )- S -7-(adamant-1-ylamino)-6-(tert-butoxycarbonyl)-7-oxoheptyl-2-methylpropanethioate), a selective HDAC6 inhibitor ; ( B ) Real-time PCR assay for K Ca 1.1 in MDA-MB-453 cells transfected with control siRNA (si-ctrl) and siRNAs specific for HDAC2 and HDAC3 (siHDAC2, siHDAC3) for 48 h ( n = 4 for each). Expression levels were expressed as a ratio to ACTB. Results are expressed as means ± SEM ( n = 4 for each). * p < 0.05; ** p < 0.01 vs. the vehicle control or control siRNA-transfected group.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal:

Article Title:

doi:

Figure Lengend Snippet: Effects of treatments with VD agonists on expression levels of HDAC2 transcripts and proteins in MDA-MB-453 cells. ( A ) Real-time PCR assay for HDAC2 in VD agonist-treated MDA-MB-453 cells for 72 h ( n = 4 for each). Expression levels were expressed as a ratio to ACTB; ( B ) Protein lysates of VD agonist-treated MDA-MB-453 cells were probed by immunoblotting with anti-HDAC2 (upper panel) and anti-ACTB (lower panel) antibodies on the same filter; ( C ) Summarized results are obtained as the optical density of HDAC2 and ACTB band signals in B . After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the HDAC2 signal in the vehicle control was expressed as 1.0 (dotted line, n = 4 for each). Results are expressed as means ± SEM ( n = 4 for each). ** p < 0.01 vs. the vehicle control.

Article Snippet: In the immunocytochemical examination, MDA-MB-453 cells were harvested using a sterile cell scraper, and non-permeabilized cells were stained using a rabbit polyclonal K Ca 1.1 (extracellular) antibody (APC-151, Alomone Labs) plus Alexa Fluor ® 488-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot