Journal: Acta Histochemica et Cytochemica

Article Title: Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca 2+ Channels in Rat Adrenal Medullary Chromaffin Cells

doi: 10.1267/ahc.23-00042

Figure Lengend Snippet: Different distribution of Ca V 1.2 channel and muscarinic M 4 receptor in the cell membrane in rat AMC cells. ( A ) Immunostaining of Ca V 1.2-YFP fusion protein expressed in HEK293T cells with a rabbit anti-Ca V 1.2 antibody (Ab). HEK293T transfected with a Ca V 1.2-YFP construct were labeled with the rabbit anti-Ca V 1.2 Ab. a and b represent confocal images of Ca V 1.2-like immunofluorescence and YFP fluorescence, respectively; c represents a differential interference contrast (DIC) image. The immunoreaction and YFP fluorescence were visualized with excitation at 514 nm and emission of 530–600 nm and with excitation at 633 and emission above 650 nm, respectively. ( B ) Fractionation analysis of rat adrenal medullae for integral membrane proteins. The cell membrane was divided into the raft and non-raft membrane domains by using discontinuous sucrose density gradient centrifugation (see the Materials and Methods). The same volume of each fraction with 5%–40% sucrose was immunoblotted for caveolin-1, transferrin receptor (R), muscarinic M 4 receptor, and TASK1 channel. Note that caveolin-1, a raft membrane marker, was enriched in the 20% fraction, whereas transferrin R, a non-raft membrane marker, was present in the 40% fraction. ( C ) Double staining for caveolin-1 and Ca V 1.2 and for M 4 receptor and Ca V 1.2 in rat AMC cells. The first column indicates confocal images of caveolin-1 and M 4 receptor-like immunofluorescence. The second column shows confocal images of Ca V 1.2-like immunofluorescence. The third column is a merge of immunofluorescence images. The fourth column shows DIC images. The calibration applies to all the images. Dissociated rat AMC cells were treated overnight with rabbit anti-Ca V 1.2 Ab (dilution, 1:50) and mouse anti-caveolin-1 Ab (1:20) or mouse anti-M 4 Ab (1:50). Ca V 1.2 and caveolin-1 or M 4 receptor-like immunoreactive material were visible as rhodamine and FITC-like fluorescence, respectively. ( D ) Summary of the coincidence rates of caveolin-1 (Cav1) and M 4 with Ca V 1.2. The data represent the mean ± SEM (Cav1/Ca V 1.2, n = 10; M 4 /Ca V 1.2, n = 5). Statistical significance was evaluated with an unpaired Student’s t test.

Article Snippet: First, it was incubated with one of the following primary antibodies (Abs): rabbit anti-caveolin-1 (sc-894: Santa Cruz Biotechnology, Santa Cruz, CA, USA) (RRID:AB_2072042), mouse anti-transferrin receptor (A11130: Molecular Probes, Eugene, OR, USA) (RRID:AB_2534136), mouse anti-M 4 (MAB1576: Chemicon, Temecula, CA, USA) (RRID:AB_2080217), rabbit anti-Ca V 1.2 (ACC-001: Alomone, Jerusalem, Israel) (RRID:AB_2039764), or rabbit anti-TWIK-related acid-sensitive K + 1 (TASK1) (APC-024: Alomone) (RRID:AB_2040132).

Techniques: Membrane, Immunostaining, Transfection, Construct, Labeling, Immunofluorescence, Fluorescence, Fractionation, Gradient Centrifugation, Marker, Double Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca 2+ Channels in Rat Adrenal Medullary Chromaffin Cells

doi: 10.1267/ahc.23-00042

Figure Lengend Snippet: Diagram showing localization of caveolin-1, Ca V 1.2, muscarinic M 4 receptor subtype, and TASK1 in the raft and non-raft membrane domains.

Article Snippet: First, it was incubated with one of the following primary antibodies (Abs): rabbit anti-caveolin-1 (sc-894: Santa Cruz Biotechnology, Santa Cruz, CA, USA) (RRID:AB_2072042), mouse anti-transferrin receptor (A11130: Molecular Probes, Eugene, OR, USA) (RRID:AB_2534136), mouse anti-M 4 (MAB1576: Chemicon, Temecula, CA, USA) (RRID:AB_2080217), rabbit anti-Ca V 1.2 (ACC-001: Alomone, Jerusalem, Israel) (RRID:AB_2039764), or rabbit anti-TWIK-related acid-sensitive K + 1 (TASK1) (APC-024: Alomone) (RRID:AB_2040132).

Techniques: Membrane

Journal: Scientific Reports

Article Title: Stat3 as a potential therapeutic target for rheumatoid arthritis

doi: 10.1038/s41598-017-11233-w

Figure Lengend Snippet: Stat3 is required for inflammation and osteoclast activation in CIA models. ( a–f ) CIA was induced by collagen injection in 5-week-old control (Ctl) or Stat3 cKO mice, and mice were administered PolyIpolyC (1.25 µg/kg/day) IP on days -21, -20, -19, -14 and -7 before a second type II collagen with CFA injection. Serum IL-6 ( c ) and IL-17 ( d ) protein levels in Ctl or Stat3 cKO mice were assessed by ELISA 14 days after the second injection. Specimens of ankle joint tissues from CIA Ctl or Stat3 cKO mice were subjected to immunofluorescence staining using pStat3 ( a ) or Cathepsin K ( e ) antibodies. Nuclei were stained with DAPI. Bar, 100 µm. Data in ( c ) represent mean IL-6 (pg/ml) ± SD ( n = 8 for control, n = 7 for Stat3 cKO mice, * P < 0.05). Data in ( d ) represent mean and IL-17 (pg/ml) ± SD ( n = 5 for control, n = 6 for Stat3 cKO mice, * P < 0.05). Data in ( b ) and ( f ) represent mean relative areas ± SD of Stat3-positive ( b ) and Cathepsin K-positive ( f ) cells, respectively ( n = 3 for control, n = 3 for Stat3 cKO mice, * P < 0.05).

Article Snippet: After blocking with 3% BSA in PBS for 1 h, sections were stained for 6 h with rabbit anti-mouse pSTAT3 (1:100 dilution; Cell Signaling Techniques, Inc.) or rabbit anti-mouse Cathepsin K (1:100 dilution; Abcam) at 4 °C.

Techniques: Activation Assay, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Stat3 as a potential therapeutic target for rheumatoid arthritis

doi: 10.1038/s41598-017-11233-w

Figure Lengend Snippet: Effect of Stat3 inhibitors on Stat3 activation and osteoclast formation in CIA models. ( a – d ) Arthritis was induced in 5-week-old wild-type DBA/1 J male mice by injection of type II collagen and CFA on day -21 followed by the second injection on day 0. Vehicle, CP690550 or meloxicam (each 15 mg/kg/day) was administered IP once a day for 2 weeks starting at day 0. Specimens of ankle joints derived from CIA mice administered indicated drugs were subjected to immunofluorescence staining for pStat3 ( a ) or Cathepsin K 14 days after the second injection ( c ). Nuclei were visualized by DAPI. Bar, 100 µm. Data in ( b ) and ( d ) represent mean relative areas ± SD of Stat3-positive ( b ) and Cathespsin K-positive ( d ) cells, respectively ( n = 3 each, ** P < 0.01). ( e ) Total RNA was prepared from NIH3T3 cells treated 8 h with or without IL-6 (100 ng/ml) plus sIL-6R (100 ng/ml) in the presence or absence of vehicle, CP690550 or Meloxicam (1 μM). RANKL expression was then analyzed by realtime PCR. Data represents mean RANKL expression relative to β actin ± SD ( n = 3, * P < 0.05, NS not significant). ( f ) M-CSF-dependent wild-type bone marrow cells (1 × 10 5 cells per well) were cultured with osteoblastic MC3T3E1 cells (1 × 10 4 cells per well) plus IL-6 (100 ng/ml) and sIL-6R (100 ng/ml) in the presence or absence of indicated drugs (1 μM each). Eight days later, mRNA was harvested for osteoclastic marker analysis. Representatives of at least two independent experiments are shown.

Article Snippet: After blocking with 3% BSA in PBS for 1 h, sections were stained for 6 h with rabbit anti-mouse pSTAT3 (1:100 dilution; Cell Signaling Techniques, Inc.) or rabbit anti-mouse Cathepsin K (1:100 dilution; Abcam) at 4 °C.

Techniques: Activation Assay, Injection, Derivative Assay, Immunofluorescence, Staining, Expressing, Cell Culture, Marker