Brains were collected in liquid nitrogen and lysed in TNE buffer on ice. Protein content was quantified with the BCA Protein Assay Kit (Thermo Fisher Scientific). Then, 10-μg protein samples were electrophoresed and transferred to PVDF membranes (Merck Millipore, Burlington, MA, USA). After 1 h of blocking with 5% non-fat dry milk, membranes were incubated with primary antibody overnight at 4 °C. After 1 h of secondary antibody incubation at room temperature, visualization was conducted with a luminoimage analyzer (ImageQuant LAS4000, GE Healthcare). For RBFox-1 normalization, membranes were subjected to WB Stripping solution (nacalai, Kyoto, Japan) after chemiluminescent exposure, then incubated with primary anti-β-actin antibody overnight at 4 °C. The gray value of each lane was measured with Image-J software, and normalized to β-actin to obtain protein expression. The following antibodies were used: CRTC1 (1:1000; Cell Signaling Technology, Danvers, MA, USA); TJAP-1 (1:1000; Novus Biologicals); RBFox-1 (1:1000; Merck Millipore); E-Cadherin (1:1000; Cell Signaling Technology); Claudin-1 (1:1000; Invitrogen); cleaved Caspase-3 (1:1000; Cell Signaling Technology); β-actin (1:5000; Proteintech, Rosemont, IL, USA).
Crtc1
Manufactured by Cell Signaling Technology
Sourced in United States
CRTC1 is a laboratory reagent that is a transcriptional coactivator. It functions to regulate gene expression in response to cellular signals.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Wb stripping solution, by Nacalai Tesque (1 mentions)
Claudin 1, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Lsm 700 microscope, by Zeiss (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Odyssey system, by LI COR (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Histone h1, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Sox10, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 protocols using crtc1
1
Western Blot Analysis of Neurological Markers
2
Immunohistochemical Analysis of HCN2 and CRTC1 in Mouse Brain
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Immediately after the behavioral test, mice received overdose anesthesia with pentobarbital and were subsequently perfused with PBS and fixed by 4% PFA. The brain tissues were then sectioned into 20‐μm‐thick cryosections for the evaluation of HCN2 and CRTC1, as previously described.
30 (link) Briefly, nonspecific component was removed with a blocking buffer containing 0.3% Triton X‐100, 1% BSA, and 5% goat serum. The blocked sections were incubated first with primary antibodies against CRTC1 (1:200, Cell Signaling Technology, USA), HCN2 (1:200, Alomone), or NeuN (1:500, Millipore) overnight at 4°C, and then with the secondary antibody (anti‐rabbit Alexa Fluor 488 conjugated, diluted at 1:800) for 1 h at room temperature. The staining of mPFC sections was captured using a Zeiss LSM 700 microscope.
30 (link) Briefly, nonspecific component was removed with a blocking buffer containing 0.3% Triton X‐100, 1% BSA, and 5% goat serum. The blocked sections were incubated first with primary antibodies against CRTC1 (1:200, Cell Signaling Technology, USA), HCN2 (1:200, Alomone), or NeuN (1:500, Millipore) overnight at 4°C, and then with the secondary antibody (anti‐rabbit Alexa Fluor 488 conjugated, diluted at 1:800) for 1 h at room temperature. The staining of mPFC sections was captured using a Zeiss LSM 700 microscope.
3
Western Blot Analysis of CRTC1, GAPDH, FLAG, and HA
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50 μg brain tissues were homogenized in 300 μl RIPA buffer containing protease and phosphatase inhibitor. After grinding 2 min, tissues were lysed for 30 min at 4°C. Total protein concentration was quantified using the BCA assay. Equivalent amounts of proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h, and incubated overnight at 4°C with primary antibodies: CRTC1 (Cell Signaling Technology, United States, dilution: 1:1,000), GAPDH (Cell Signaling Technology, United States, dilution: 1:1,000), FLAG (Sigma, United States, dilution 1:1,000) and HA (Cell Signaling Technology, United States, dilution: 1:1,000). Then membranes were incubated with anti-rabbit secondary antibody for 2 h and detected by Odyssey system (LI-COR Biosciences).
4
Western Blot Analysis of Melanogenic Proteins
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Protein extracts were prepared with RIPA buffer (iNtRON, IBS-BR001), resolved on SDS-acrylamide gels by electrophoresis and transferred to polyvinylidene difluoride membrane (Roche, 03010040001). Western blots were blocked with either 5% non-fat milk (Becton-Dickinson, 23100) or 5% bovine serum albumin (BSA, Affymetrix, 10857) in Tris-buffered saline containing 0.05% Tween 20 (Sigma-Aldrich, P1379). After washing, blots were reacted with primary antibody at 4℃ overnight followed by secondary antibody for 1 h at room temperature. Immune complex on the blots was visualized by reacting with enhanced chemiluminescence reagent (GE Healthcare, RPN2232). This study employed primary antibodies against MITF-M (Abcam, ab12039), TYR (Santa Cruz, sc-7833), p-CREB (Cell Signaling, 9198), CREB (Cell Signaling, 9197), p-CRTC1 (Cell Signaling, 3359), CRTC1 (Cell Signaling, 2587), 14-3-3 (Santa Cruz, sc-1657), p-β-catenin (Cell Signaling, 9567), β-catenin (Cell Signaling, 9562), SOX10 (Santa Cruz, sc-17342), GAPDH (Santa Cruz, sc-25778) or histone H1 (Santa Cruz, sc-8030). Secondary antibodies were rabbit anti-goat IgG labeled with horseradish peroxidase (HRP) (Thermo Fisher Scientific, A27011), goat anti-rabbit IgG labeled with HRP (Thermo Fisher Scientific, 31460), and goat anti-mouse IgG labeled with HRP (Thermo Fisher Scientific, 31430).
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