primary antibodies against ccr5  (PeproTech)

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Western blot analysis of CCRs protein in PC12 cell lines. (a) Approximately 40–60 μ g of total protein was resolved on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose membranes. Membranes were probed with polyclonal anti-CCR1, polyclonal anti-CCR3, or monoclonal anti-CCR5. Representative blots are shown and arrows indicate the main band of the receptors with apparent MW 41 kDa for CCR1, 40 kDa for CCR3, and 46 kDa for CCR5. (b) The bands intensity was densitometrically analyzed and the results are expressed as % of the control PC12 cells obtained after normalization to endogenous β -actin level (± SD). ∗ P < 0.05 (n=7), C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Western Blot, SDS Page, Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Localization of CCR5 in PC12 cell lines. PC12 cells differentiated for 48 h with db-cAMP were fixed and immunostained with antibodies against CCR5 receptor (green) and Na + /K + ATPase (plasma membrane marker, red). Nuclei were stained with Hoechst 33342 (blue). Representative confocal images are presented. Values shown in merged images represent average fluorescence intensity ± SEM (n = 5) of pixels positive in green channel (CCR5) that colocalize with red channel positive pixels (Na + /K + ATPase). ∗ P< 0.05, ∗∗ P < 0.01. Scale bars: 10 μ m. C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Clinical Proteomics, Membrane, Marker, Staining, Fluorescence, Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Analysis of CCL5 effect on calcium transients in PC12 cell lines. Ca 2+ transients were measured in parallel wells with (red lines) or without (black lines) presence of specific inhibitors. (a) SERCA inhibitor, 1 μ M thapsigargin, was added after 50 s (red arrow), and next 50 ng/ml CCL5 was applied after 150 s (black arrow). (b) CCRs inhibitors, 1 nM BX513 for CCR1, 1 μ M SB328437 for CCR3, and 1 nM DAPTA for CCR5, were included just before measurements. 50 ng/ml CCL5 was applied after 150 s (black arrow). All measurements were done in duplicate and the presented traces are average from 5 independent cell cultures (n=10). C: control line, _2: PMCA2-reduced line, _3: PMCA3-reduced line.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Control

Journal: BioMed Research International

Article Title: Calcium Dyshomeostasis Alters CCL5 Signaling in Differentiated PC12 Cells

doi: 10.1155/2019/9616248

Figure Lengend Snippet: Schematic presentation of CCL5 effect on PC12 cells. Downregulation of neurospecific PMCA2 or PMCA3 isoforms in differentiated PC12 cells generated two types of cell response: similar for both lines or characteristic for only one line. The common changes were increased cytosolic Ca 2+ and, as a compensatory mechanism, upregulation of PMCA1 isoform, enlarged expression of SERCA2 and SERCA3, and diminished calmodulin amount [ , ]. The levels of CCR5 and IP 3 R-3 proteins also increased, but the expression of IP 3 R-1 and IP 3 R-2 was lowered (present study). Interestingly, altered IP 3 R isoform composition did not change total IP 3 R protein in the _2 line, while it increased in the _3 line. Also in _3 cells, the amount of PMCA4 increased . These subtle differences could have profound consequences after CCL5/CCR5 activation, since potency to restore the basal Ca 2+ level in the _3 line appears to be higher than in the _2 line, which may be essential for the survival of the cell. Under prolonged Ca 2+ signal in the _2 line due to reduction of the fastest isoform - PMCA2, the subsequent Ca 2+ -mediated processes could increase vulnerability to cell death. Abbreviations used: CaM, calmodulin; CCL5, chemokine C-C motif ligand 5; CCR5, receptor for CCL5; inositol 1,4,5-triphosphate (IP 3 ); IP 3 R, IP 3 receptor; PMCA, plasma membrane Ca 2+ -ATPase; SERCA, sarco/endoplasmic Ca 2+ -ATPase.

Article Snippet: Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer (10% normal goat serum, 0.1% Triton X-100, PBS pH = 7.4), following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer (1:100, Santa Cruz Biotechnology).

Techniques: Generated, Expressing, Activation Assay, Clinical Proteomics, Membrane