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).
Ab32096
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab32096 is a laboratory equipment product manufactured by Abcam. It is designed to perform a specific function in a laboratory setting. The core function of this product is to facilitate a particular experimental or analytical procedure. No further details or interpretation about the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab32515, by Abcam (23 mentions)
Ab108319, by Abcam (6 mentions)
Ab31387, by Abcam (5 mentions)
Ab181602, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Ab171095, by Abcam (3 mentions)
Ab75991, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab65783, by Abcam (2 mentions)
Ab203063, by Abcam (2 mentions)
Nb110 61576ss, by Novus Biologicals (2 mentions)
Bicinchoninic acid bca assay kit, by Beyotime (2 mentions)
Loading buffer, by Beyotime (2 mentions)
Ab184699, by Abcam (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Ab64693, by Abcam (2 mentions)
51 protocols using ab32096
1
Quantifying Protein Expression via Western Blot
2
Immunohistochemical Analysis of Lung Tissue
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Paraffin-embedded sections of lung tissue were assessed with IHC. Endogenous peroxidases were quenched with 0.3% H2O2, and antigen retrieval was performed using a high-pressure method on dewaxed tissue sections. Samples were then incubated with primary antibodies against α-SMA (ab32575, Eptomics, Burlingame, CA) and p-CREB (ab32096, Abcam) overnight at 4 °C, followed by incubation with a secondary antibody (PV-6000, Beijing Zhongshan Jinqiao Biotechnology Co. Ltd, China) at 37 °C for 20 min. Immunoreactivity was visualized with DAB (ZLI-9018, ZSGB-BIO, Beijing, China). Brown staining was considered positive.
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 Muscle Proteins
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Western blot analysis was performed as previously described.66 (link) The primary antibodies used were anti-FLAG (AF519, Beyotime, 1:1,000), anti-MYOD (ABP53067, Abbkine, 1:500), anti-MyHC (B103, DHSB, 0.5 μg/mL), anti-SERCA2 (NB100-237, Novus, 1:2,000), anti-ACC (PA5-17564, Thermo Fisher Scientific, 1:1,000), anti-pACCSer80 (orb315750, Biorbyt, 1:500), anti-CPT1 (bs-23779R, Bioss, 1:500), anti-pmTORSer2488 (#5536, CST, 1:1,000), anti-mTOR (bs-1992R, Bioss, 1:500), anti-ULK1 (bs-3602R, Bioss, 1:500), anti-LC3B (NB100-2220, Novus, 2.0 μg/mL), anti-P62 (18420-1-AP, Proteintech, 1:1,000), anti-CREB (bs-0035R, Bioss, 1:500), anti-pCREBSer133 (ab32096, Abcam, 1:5,000), anti-AMPK (bs-1115R, Bioss, 1:500), anti-pAMPKser712 (ABN-PAB12602, Abnova, 1:2,000), anti-Calcineurin (#2614S, CST, 1:1,000), anti-PGC-1α (66369-1-Ig, Proteintech, 1:5,000), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000). ProteinFind goat anti-mouse IgG(H+L), HRP conjugate (HS201-01, TransGen, 1:1,000) and ProteinFind goat anti-rabbit IgG(H+L), HRP conjugate (HS101-01, TransGen, 1:500) were used as secondary antibodies.
7
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).
8
Western Blot Analysis of CREB and FtMt
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OVCAR3 and SKOV3 cells transfected with shRNA-CREB or negative control were treated with Roflumilast and/or H89, and then the cells were harvested and, according to the manufacturer's instructions, the whole cell protein extracts were resolved on a 10% SDS denatured polyacrylamide gel and were then transferred onto a nitrocellulose membrane, which were blocked in 5% BSA in TBST buffer (Tris Buffer Saline containing 0.1% Tween-20) for 1 h at room temperature, and subsequently incubated with Anti-GAPDH (#ab8245, Abcam, USA), FtMt (#ab124889, Abcam, USA), CREB (#ab32096, Abcam, USA), p-CREB (#9198, Cell Signaling Technology, USA) antibodies overnight at 4°C. GAPDH was used as the loading control in the Western blotting. After washing with TBST buffer, the blots were then incubated with HRP-conjugated secondary antibody (#BS12478, #BS13278, Bioworld, China) for 1 h at room temperature. After washing with TBST buffer, the blots were visualized using the ECL-Plus reagent (Millipore, Billerica, MA, USA).
9
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.
10
CREB1 Phosphorylation Signaling Pathway
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LPS was purchased from Sigma (St Louis, USA). Antibodies against CREB1 (ab31387) and phosphorylated CREB1 (ab32096) were purchased from Abcam (Cambridge, USA). Antibody against DUOX2 (NB110-61576ss) was purchased from NOVUS (Centennial, USA). Specific inhibitors of CREB1, KG-501 (HY-103299) and 666-15 (HY-101120) were purchased from MedChem Express (MCE, Monmouth Junction, USA), Lipofectamine RNAiMAX was purchased from Thermo Fisher Scientific (Rockford, USA). Bicinchoninic acid assay (BCA) kit, RIPA lysis buffer, and loading buffer were purchased from Beyotime (Shanghai, China).
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