Western blot analysis was performed as previously described (4) (link). Briefly, protein-transferred membranes were blocked and incubated with primary antibodies (anti-RGS4, 1：500, SC-6204; anti-GABAB2R, 1：100, SC-28792; anti-Gαi-3, 1：500, SC-262; Santa Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were detected with an enhanced chemiluminescence detection kit (Amersham Biosciences, Munich, Germany) according to the manufacturer’s protocol. For quantification of the results, each band density was read by SigmaGel software (Sigma). Each density was normalized using the corresponding α-tubulin density as an internal control.
Sigmagel software
Manufactured by Merck Group
Sourced in United States
SigmaGel software is a data analysis and visualization tool designed for use with laboratory equipment. The software provides functions for organizing, analyzing, and presenting data collected from various laboratory instruments.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Cytiva (2 mentions)
Ab128915, by Abcam (1 mentions)
Sc 8002, by Santa Cruz Biotechnology (1 mentions)
Ab40755, by Abcam (1 mentions)
Sc 373853, by Santa Cruz Biotechnology (1 mentions)
Chef genomic dna plug kit, by Bio-Rad (1 mentions)
Chef drii apparatus, by Bio-Rad (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
4 protocols using sigmagel software
1
Western Blot Analysis of RGS4, GABAB2R, and Gαi-3
2
Western Blot Analysis of Protein Signaling
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Western blot analysis was conducted as previously described [31 (link)]. Proteins (10 µg each) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Protein blocking was performed with 1% bovine serum albumin and 5% skim milk. Membranes were incubated with PI3K (1: 1000, ab40755, Abcam, Cambridge, UK), Akt (1:1000, 9272S, Cell Signaling, Danvers, MA, USA), pAkt (1:1000, 9271S, Cell Signaling, Danvers, MA, USA), ER-alpha (1:1000, SC-8002, Santa Cruz, Dallas, TX, USA), ER-beta (1:1000, SC-373853, Santa-Cruz, Dallas, TX, USA), and GAPDH (1:10,000, ab128915, Abcam, Cambridge, UK) primary antibodies. Antibody interactions were visualized with an enhanced chemiluminescence detection kit (Amersham Biosciences, Munich, Germany). The density for each band was quantified by Sigma Gel software (Sigma-Aldrich, St. Louis, MO, USA) to analyze the results. Each density was normalized using the corresponding GAPDH as an internal control.
3
Yeast Chromosome Separation by PFGE
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Chromosomal DNA was isolated from the remaining 5 mL of yeast cells solutions, prepared as described in protein preparation. DNA isolation was conducted using a CHEF Genomic DNA Plug kit (Bio-Rad, Hercules, CA, USA) according to the methods described by Schwartz and Cantor [37 (link)]. Chromosomes were separated by pulsed field gel electrophoresis (PFGE) in 0.8% agarose gel by means of a CHEF-DR II apparatus (Bio-Rad). Electrophoresis was performed in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, and 10 mM EDTA; pH 8.2) at 12 °C, and the pulses were as follows: 120 s for 24 h and 240 to 360 s for 24 h, all at 4.5 V/cm. Separated chromosomes were stained in ethidium bromide (0.5 µg/mL) for 15 min with gentle agitation. The molecular weight of bands was estimated using SigmaGel software (Sigma-Aldrich, Gillingham, UK).
4
Quantitative RT-PCR Analysis of Ovarian Angiogenesis
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Total RNA was isolated from ovarian tissues with TRizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. One microgram of total RNA was reverse-transcribed using Super Script III RT (Invitrogen), random hexamers (50 pmol), and deoxynucleotide (2.5 mM) at 37°C for 20 minutes. Specific primer sequences for VEGF-A, Angpt-1, and Angpt-2 were obtained from the Gene Bank Database (Table 1 ). The PCR reactions were performed as follows: specimens were first heated to 94°C for 4 minutes, then subjected to 30 cycles of denaturation at 94°C for 40 seconds, annealed at 55°C for 40 seconds, extended at 72°C for 40 seconds, and then underwent a final elongation step at 72°C for 8 minutes. The PCR products were analyzed by 2% agarose gel electrophoresis. Expression of each mRNA species was normalized to that of glyceraldehyde-3-phosphate dehydrogenase. Relative quantitation of the target gene expression was performed using SigmaGel software (Sigma, St. Louis, MO, USA).
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