We lysed the cells with sample buffer (62.5 mM Tris-HCl buffer [pH 6.8] containing 2% sodium dodecyl sulfate [SDS], 5% β-mercaptoethanol, and 10% glycerol). The cell lysates were incubated at 95 °C and analyzed by SDS-polyacrylamide gel electrophoresis. Subsequently, immunoblotting was performed using anti-TRPV4 (#ACC-034, Alomone labs, Jerusalem, Israel) and α-tubulin (#sc-32293, Santa Cruz Biotechnology, CA, USA) antibodies. The membranes were washed and incubated with HRP-conjugated secondary antibody (for TRPV4: #sc-2004 or for α-tubulin: sc-2005, Santa Cruz Biotechnology) for 1 h at room temperature and visualized with ECL prime (GE Healthcare, Buckinghamshire, UK). The signals were detected and quantified using LAS-1000 pro (Fuji Film, Tokyo, Japan) with Image Gauge software (Fuji Film). The expression of TRPV4 was normalized to α-tubulin protein levels.
Las 1000 pro
Manufactured by Fujifilm
Sourced in Japan
The LAS-1000 pro is a digital imaging system designed for scientific and industrial applications. It features a high-resolution camera, advanced image analysis software, and a range of accessories for capturing, analyzing, and processing various types of images.
Automatically generated - may contain errors
Lab products found in correlation
Image gauge software, by Fujifilm (4 mentions)
Ecl prime, by GE Healthcare (3 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Anti nestin, by Merck Group (2 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Nbp1 04676, by Novus Biologicals (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Anti β tubulin 3, by Merck Group (1 mentions)
4 protocols using las 1000 pro
1
TRPV4 Protein Expression Analysis
2
Quantification of Nestin Expression
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Whole-cell lysates were extracted with lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 5% β-mercaptoethanol, and 10% glycerol), and the protein concentration was measured using Bradford ULTRA (Novexin, Cambridge, UK). A total of 5 μg of protein was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% non-fat milk for 30 min, the membrane was incubated overnight at 4 °C with anti-nestin (1:1000; Millipore, CA, USA) and anti-HSP90 (1:1000; Santa Cruz Biotechnology, CA, USA) antibodies. Membranes were washed and incubated with HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology) for 1 h at room temperature and visualized with ECL prime (GE Healthcare, Buckinghamshire, UK). The chemiluminescent signals were detected and quantified using LAS-1000 pro (Fuji Film, Tokyo, Japan) with Image Gauge software (Fuji Film). HSP90 was used as an internal control. To normalize the nestin expression, the chemiluminescent signal of nestin was divided by the chemiluminescent signal of HSP90.
3
Protein Expression Analysis in Neuron Cultures
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DNs were lysed with sample buffer (62.5 mM Tris–HCl buffer [pH 6.8] containing 2% sodium dodecyl sulfate [SDS], 5% β‐mercaptoethanol, and 10% glycerol) and incubated at 95°C for 5 min. The proteins in the cell lysates were electrophoresed using SDS‐polyacrylamide gel electrophoresis, and immunoblotting was performed using rabbit anti‐nuclear receptor‐related 1 (NURR1; #10975‐2‐AP; Proteintech), mouse anti‐TH (#66334‐1‐Ig; Proteintech), rabbit anti‐forkhead box protein A2 (FOXA2; #22474‐1‐AP; Proteintech), rabbit anti‐peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α; #NBP1‐04676; Novus Biologicals), mouse anti‐postsynaptic density protein 95 (PSD‐95; #MAB1598; Millipore), rabbit anti‐paired‐like homeodomain 3 (PITX3; #CSB‐PA010844LA01HU; CUSABIO), mouse anti‐microtubule‐associated protein 2 (MAP2; #M9942; Sigma–Aldrich), rabbit anti‐delta like non‐canonical Notch ligand 1 (DLK1; #10636‐1‐AP; Proteintech), rabbit anti‐DAT1; #22524‐1‐AP; Proteintech) and mouse anti‐α‐tubulin (#sc‐32,293; Santa Cruz Biotechnology) antibodies. The immunoreactive bands were detected using ECL Prime (Cytiva) and analyzed using LAS‐1000 pro (Fuji Film) and Image Gauge software (Fuji Film). α‐tubulin was used as an internal control. To normalize the protein expression, the chemiluminescent signal was divided by the chemiluminescent signal of α‐tubulin.
4
Protein Expression Analysis of Cell Lysates
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The cells were washed three times with PBS and then lysed with sample buffer (62.5 mM Tris-HCl buffer [pH 6.8] containing 2% sodium dodecyl sulfate [SDS], 5% β-mercaptoethanol, and 10% glycerol). The cell lysates were incubated for 95°C and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Subsequently, immunoblotting was performed using anti-β-tubulin III (Sigma-Aldrich), anti-Nestin (Millipore), anti-Tom20 (Santa Cruz H. Kato et al.
Biotechnology), and anti-heat shock protein 90 (HSP90; Santa Cruz Biotechnology) antibodies. The immunoreactive bands were detected using ECL Prime (GE Healthcare, Buckinghamshire, UK) and analyzed using LAS-1000 pro (Fuji Film, Tokyo, Japan) and Image Gauge software (Fuji Film) .
Biotechnology), and anti-heat shock protein 90 (HSP90; Santa Cruz Biotechnology) antibodies. The immunoreactive bands were detected using ECL Prime (GE Healthcare, Buckinghamshire, UK) and analyzed using LAS-1000 pro (Fuji Film, Tokyo, Japan) and Image Gauge software (Fuji Film) .
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