The total protein was extracted using a radioimmunoprecipitation assay, and the bicinchoninic acid (Beyotime, Shanghai, China) method was employed to quantify the concentration. The protein sample was first electrophoresed for 2h; subsequently, the authors transferred the total protein onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). TBST containing 5% skim milk was used to block non-specific antigens for 1h following incubating with primary antibodies (caspase-3 [1:1,000, ab32351; Abcam], c-caspase-3 [1:1,000, ab32042; Abcam], CREB1 [1:1,000, ab32515; Abcam], p-CREB1 [1:1,000, ab32096; Abcam], Bcl-2 [1:1,000, ab182858; Abcam], PSD-95 [1:1,000, ab238135; Abcam], synaptophysin [1:1,000, ab32127; Abcam], synapsin [1:1,000, ab254349; Abcam], GAPDH [1:1,000, ab8245; Abcam], and β-actin [1:5,000, #A5441, Sigma]) at 4°C overnight. Membranes were washed with TBST three times and incubated with the secondary HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution; cat. ab205719; Abcam). Subsequently, membranes were washed with TBST three times. Blots were then visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK).
Ab32515
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab32515 is a lab equipment product manufactured by Abcam. It is a scientific instrument designed for use in research and laboratory settings. The core function of this product is to perform a specific task or measurement, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab32096, by Abcam (23 mentions)
Ab8245, by Abcam (5 mentions)
Ab181602, by Abcam (5 mentions)
Ab108319, by Abcam (5 mentions)
Ab6721, by Abcam (4 mentions)
Ab182858, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Ab203063, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab17942, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ab208035, by Abcam (2 mentions)
Ab170099, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab18258, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab76238, by Abcam (2 mentions)
40 protocols using ab32515
1
Quantifying Protein Expression via Western Blot
2
ATM Signaling Pathway Evaluation
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A lymphoblastoid cell line (LCL) was derived from each patient’s blood. Immunoblotting for ATM expression and ATM activity assays were performed using methods previously described (7 (link), 8 (link)). Briefly, following activation of ATM by exposure of cells to 2-Gy x-rays, cells were harvested at 30 min and the ATM present in patient derived cells was tested for its ability to phosphorylate a group of target proteins (Smc1, KAP1, Nbn and CREB) using phosphospecific antibodies. Antibodies used for immunoblotting were ATM 11G12 (custom made mouse monoclonal), pATM Ser1981 #AF1655 (R&D Systems, Abingdon, UK), Smc1 #A300-055A, pSmc1 Ser966 #A300-050A, KAP-1 #A300-275A, pKAP-1 Ser824 #A300-767A (Bethyl Laboratories, Universal Biologicals, Cambridge, UK), Nbs1 #Ab23996, pNbs1 Ser343 #Ab47272, CREB #Ab32515 (Abcam, Cambridge, UK), pCREB Ser121 #NB100-410 (Novus Biologicals, Cambridge, UK).
An LCL derived from a normal individual and another derived from a known classical A-T patient were used as positive and negative controls for ATM activity/signalling. Patients were classified as having classical or variant A-T based on either the absence of any detectable activity/signalling or the presence of some residual ATM activity/signalling, respectively (Figure 1
An LCL derived from a normal individual and another derived from a known classical A-T patient were used as positive and negative controls for ATM activity/signalling. Patients were classified as having classical or variant A-T based on either the absence of any detectable activity/signalling or the presence of some residual ATM activity/signalling, respectively (
3
Evaluating Ca2+/CaMKII/CREB Pathway
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Western blotting was performed to determine the expression changes in the Ca2+/CaMKII/CREB pathway. The cryopreserved hippocampal tissues were added with 1 mL of protein lysis buffer containing 1% protease, homogenized, placed on ice for 30 min, and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were collected, and their protein concentration determined by the bicinchoninic acid (BCA) method. Next, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), transferred onto a membrane and sealed in skim milk for 2 h, followed by incubation with nuclear factor-kappa B (NF-κB) p65 antibody (ab16502, Abcam, USA), B-cell lymphoma-2 (Bc1-2) (ab59348, Abcam, USA), Bcl-2-associated X protein (Bax) (ab32503, Abcam, USA), NMDAR2B antibody (ab65783, Abcam, USA), CaMKII (ab22609, Abcam, USA), CREB (ab32515, Abcam, USA), p-CREB (ab32096, Abcam, USA), BDNF (ab108319, Abcam, USA), and GAPDH antibody (ab181602, Abcam, USA) at 37°C for 1 h and at 4°C overnight. The membrane was washed 3 times with TBST for 10 min and then incubated with an HRP-labeled goat anti-rabbit IgG antibody for 1 h at 37°C. After membrane washing, the proteins were reveled using ECL kit and gel imaging system, and the absorbance was analyzed by Image Tools software.
4
Western Blot Analysis of SIRT1 and CREB1
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Proteins of 40–50 μg isolated from cells or tissues were resolved by SDS/PAGE, followed by transferring to polyvinylidene difluoride membranes (PVDF; Bio-Rad, U.S.A.). The membranes were blocked with non-fat milk and then probed with antibodies against SIRT1 (ab110304, abcam), CREB1 (ab32515, abcam), p-CREB1 (ab32096, abcam), and GAPDH (ab181602, abcam). After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies. GAPDH was used as a loading control.
5
Spinal Cord Injury and CREB Expression
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At the end of the experiment, all rats were anesthetized (n = 4 rats per group), fixed with 4% paraformaldehyde, and the targeted spinal cord segment (L4–L5) was separated according to the corresponding nerve root. All the segments were put into 4% paraformaldehyde, were fixed for one night, then dehydrated with alcohol gradient, and finally embedded into paraffin. Next, these spinal cord segments were cut horizontally, and the thickness of each section was 5 μm. One was taken from every six sections and three ones were taken for each sample. After dewaxing and hydration, incubation with H2O2, microwave retrieval and serum blocking, Rabbit anti-CREB monoclonal antibody (ab32515, division 1:500, Abcam, USA), Rabbit anti-CREB (phosphe s133) monoclonal antibody (ab32096, division 1:200, Abcam, USA), Rabbit anti-GAD65 polyclonal antibody (ab203063, division 1:200, Abcam, USA), and mouse anti-GAD67 monoclonal antibody (MAB5406, division 1:1000, Minipore, Germany) were added on each slide and then incubated overnight at 4°C. The next day, after PBS washing of the pieces, the goat anti-rabbit or rabbit anti-mouse antibody labeled with biotin was added and then incubated in a 37°C incubator for 1 h, and the expression was visualized by DAB incubation. Pictures were taken by Olympus DP71 and the Image-Pro Plus 6.0 was used for optical density analysis.
6
Western Blot Analysis of Protein Expression
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The total protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with protease inhibitor and phosphatase inhibitors (Beyotime, Nanjing, China) and quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Total protein extracts (30 µg) from exosomes, cells, and submandibular gland tissue were separated via 10% SDS‑PAGE (Epizyme, Shanghai, China) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) and blocked with 5% non‑fat milk for 1 h at room temperature. After incubation with the primary antibody and the secondary antibody, the target protein was visualized by chemiluminescence using an ECL kit (Beyotime, Nanjing, China). The antibodies used in the Western blot assay are listed as follows: GAPDH (1:10,000, ab181602, Abcam), AQP5 (1:1000, sc-514022, Santa Cruz), CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab125011, Abcam), ALIX (1:1000, ab275377, Abcam), GPER (1:1000, ab260033, Abcam), CREB (1:1000, ab32515, Abcam), p-CREB (1:5000, ab32096, Abcam), PKA (1:1000, #4782, Cell Signaling Technology), p-PKA (1:1000, #4781, Cell Signaling Technology) and goat anti-rabbit IgG H&L (HRP) antibody (1:5000, #31460, Thermo Fisher Scientific).
7
Whole-Cell Lysis and Protein Analysis
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Whole-cell lysis assay (KeyGEN) was applied for extracting protein from SMSCs and BCA protein assay kit (Biyuntime, China) assessed protein concentration. Protein (20 μg) is transferred to a PVDF membrane by SDS-polyacrylamide gel electrophoresis. Primer antibody including anti-Runx2 (Abcam, 1:1 000, ab23981), anti-PTH1R (Abcam, 1:1 000, ab75150), anti-pCREB (Abcam, 1:1 000, ab32096), anti-CREB (Abcam, 1:1 000, ab32515), anti-pSmad2/3 (Abcam, 1:1 000, ab272332), anti-Smad2/3 (Abcam, 1:1 000, ab217553), and anti-β-actin (Santa Cruz Biotechnology, 1:1 000, CA) antibodies were incubated overnight at 4 °C. Next day, the secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1/5 000) was incubated in room temperature 1 h. ChemiDoc XRS + system (BD, Franklin Lakes, NJ) was used to detect the immunoreactive bands.
8
Western Blot Analysis of Hippocampal BDNF-TrkB Signaling
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Western blot analysis was performed as described previously.
7 (link),
24 (link) The hippocampal tissues were collected at 1, 2, 3, 7, and 14 days after HI insults and immediately placed on ice. The tissues were then homogenized with a protease and phosphatase‐supplemented lysis buffer. Next, by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, equal amounts of proteins (40 μg/well) were separated and transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: anti‐BDNF (ab108319, Abcam), anti‐TrkB (phospho Y705) (ab229908, Abcam), anti‐TrkB (ab187041, Abcam), anti‐CREB (phospho S133) (ab32096, Abcam), anti‐CREB (ab32515, Abcam) and anti‐β‐actin (ab8227, Abcam). The following day, the membranes were incubated at room temperature for 2 h with the secondary antibodies (ab205718, Abcam). We visualized and photographed protein bands using enhanced chemiluminescence substrate kits (ab133406, Abcam) and a GE Amersham Imager 600 (AI600; GE Healthcare). Full unedited gel/blot from this study can be found in DataS1 .
7 (link),
24 (link) The hippocampal tissues were collected at 1, 2, 3, 7, and 14 days after HI insults and immediately placed on ice. The tissues were then homogenized with a protease and phosphatase‐supplemented lysis buffer. Next, by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, equal amounts of proteins (40 μg/well) were separated and transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: anti‐BDNF (ab108319, Abcam), anti‐TrkB (phospho Y705) (ab229908, Abcam), anti‐TrkB (ab187041, Abcam), anti‐CREB (phospho S133) (ab32096, Abcam), anti‐CREB (ab32515, Abcam) and anti‐β‐actin (ab8227, Abcam). The following day, the membranes were incubated at room temperature for 2 h with the secondary antibodies (ab205718, Abcam). We visualized and photographed protein bands using enhanced chemiluminescence substrate kits (ab133406, Abcam) and a GE Amersham Imager 600 (AI600; GE Healthcare). Full unedited gel/blot from this study can be found in Data
9
Quantification of Hippocampal and Cortical Proteins
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Western blotting was performed to determine the CREB, phosphorylated (p)‐CREB, ERK, and p‐ERK protein levels in the hippocampus and prefrontal cortex and the MT1 and MT2 receptor levels in the SCN. Briefly, the primary antibodies used for western blotting were as follows: anti‐MT1 receptor antibody (1:1000; Abcam, Cambridge, UK, ab203038), anti‐MT2 receptor antibody (1:100; Abcam, ab203346), anti‐ERK1/2 antibody (1:200; Abcam, ab17942), anti‐p‐ERK1/2 antibody (1:50; Santa Cruz Biotechnology, sc‐81492), anti‐CREB antibody (1:1000; Abcam, ab32515), anti‐p‐CREB antibody (1:1000; Abcam, ab32096), and β‐actin (1:5000; Servicebio, GB12001). Band intensities were quantified by infrared scanning densitometry (Odyssey Imaging Systems; LI‐COR Biosciences).
10
Western Blot Analysis of Phospho-CREB
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Cells were rinsed with PBS prior to adding buffer 6 (R&D Systems, Minneapolis, MN, USA) for protein isolation. Protein concentration was determined with the BCA assay (Pierce-ThermoFisher Scientific, Rockford, IL, USA). After SDS-PAGE and transfer of proteins onto a polyvinyl difluoride (PVDF) membrane; nonspecific sites were saturated with 5% milk. Incubation was performed overnight (4°C) with the following primary antibodies: anti-phospho-CREB phospho S311 (ab32096, diluted 1:500, Abcam, Cambridge, UK), anti-CREB (ab32515, diluted 1:500, Abcam, Cambridge, UK), and anti-beta-Actin rabbit mAb (#4970, diluted 1:2000, Cell Signaling Technology, Cambridge, UK). Immunodetection was performed by incubation 1 h with peroxidase-conjugated secondary antibody goat anti-rabbit IgG (#7074, diluted 1:2000, Cell Signaling Technology, Cambridge, UK), with an ECL system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The signals were quantified by densitometric scanning (Bio Rad ChemiDoc XRS+, Bio-Rad Laboratories, Inc., Hercules, USA).
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