The relative protein expressions of MRP, LRP, BCRP, Bcl2, and PKC-α in HepG2 and HepG2/DOX cells and tumor tissues were detected by western blotting. Total proteins in cells or tumor tissues were extracted by RIPA buffer (Beyotime Biotechnology, Shanghai, China), and then the protein concentration was detected using a bicinchoninic acid (BCA) assay kit (Solarbio, USA). After 20-μg protein samples were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 5% nonfat milk and washed with Tris-buffered saline containing Tween 20 (TBST). The membranes were incubated with the primary antibodies against MRP (DF8801, Affinity), LRP1 (ab92544, Abcam), BCRP (ab207732, Abcam), Bcl2 (ab196495, Abcam), PKC-α (ab32376, Abcam), and β-actin (ab8226, Abcam) overnight at 4°C. The membranes were then washed with TBST and further incubated with the horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. Finally, the protein bands were exposed to the chemiluminescent reagent (enhanced chemiluminescence) for 3 min and captured with the Chemi Capture software.
Ab32376
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32376 is a lab equipment product manufactured by Abcam. It serves as a core laboratory tool. Detailed specifications and intended applications are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab92544, by Abcam (1 mentions)
Ab207732, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Anti cyclin d2, by Santa Cruz Biotechnology (1 mentions)
Ab52617, by Abcam (1 mentions)
Ab66217, by Abcam (1 mentions)
Ab32502, by Abcam (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ab215037, by Abcam (1 mentions)
Ab254264, by Abcam (1 mentions)
Lsm700, by Leica (1 mentions)
Dm4000b, by Leica (1 mentions)
17 protocols using ab32376
1
Protein Expression Analysis in HepG2 Cells
2
Comprehensive Antibody Panel for Cell Signaling
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Mouse anti‐AKAP95 (sc‐100 643), anti‐Cx43 (sc‐13 558), anti‐cyclin D2 (sc‐53 637), anti‐cyclin D3 (sc‐6283), and anti‐Cdk2 (sc‐70 829) antibodies were purchased from Santa Cruz Biotechnology (TX, USA). Rabbit anti‐cyclin D1 (ab134175), anti‐PKB (ab8805), anti‐PKC (ab32376), anti‐ERK1/2 (ab17942), anti‐ERK5 (ab40809), anti‐AKAP95 (ab140628), anti‐cyclin E1 (ab33911), and anti‐cyclin E2 (ab40890) antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti‐cyclin D1‐T286 (3300S) antibody was purchased from Cell Signaling Technology (MA, USA). Mouse anti‐GAPDH (D190090‐O100) was purchased from BBI Life Sciences (Shanghai, China).
3
Western Blot Analysis of ABCA1, ABCG1, and PKC
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Protein was extracted from cells or tissues using RIPA buffer with protease inhibitors (Sigma). Then, 5 × protein loading buffer was added to the sample at a protein: buffer ratio of 4:1, and the sample was heated in a metal bath at 60 °C for 10 min. Protein electrophoresis was performed using the TGX FastCast acrylamide kit (BIO) and then transferred to PVDF membranes (Millipore, Bedford, MA, America). After blocking in 5% skim milk for 2 h, the membrane was incubated with primary antibody (1:1000 or 1:500) in a refrigerator at 4 °C overnight. GAPDH was used as the loading control. Next, the membrane was washed in (0.1%) (TBS-T) at least three times for 10 min each and incubated with horseradish peroxidation-enhanced secondary antibody (1:10,000) for 2 h. ECL Prime (G.E.) was used to test the protein bands according to the manufacturer's instructions. The antibodies used in the experiment were as follows: GAPDH (ProMab,20,035); ABCA1 (Abcam,ab66217); ABCG1 (Abcam,ab52617); and p-PKCα (Abcam, ab32502;PKCα (Abcam, ab32376).
4
Immunohistochemical Analysis of HCC Biomarkers
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We employed HCC tissue microarray (HLivH180Su10) from ShGnghGi Outdo Biotech company, which containing 93 paired HCC and paracancerous tissue to further confirm the relation between genes, prognosis and clinical features (detail were shown in Supplement Table 1 ). Antibody DCN (ab268048, Abcam, 1:200), NDRG1 (ab124689, Abram, 1:500), DDIT4 (ab223034 Abcam, 1:100), PRKCA (ab32376, Abcam, 1:200) were used for IHC according the instructions.
5
Immunohistochemical Detection of Phospho-ERK and PKCα in Mouse Intestinal Epithelium
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Deparaffinized sections of formaldehyde-fixed, paraffin-embedded mouse intestinal epithelium were rehydrated with ddH2O for 5 min. For antigen retrieval, slides were heated in preheated 1× target retrieval solution (DAKO) for 30 to 70 min at 90 °C, followed by cooling for 20 min. Endogenous peroxidase was inactivated with 3% H2O2. For pERK staining, sections were blocked with 5% normal goat serum in TBS-T. Sections were then incubated with primary anti-pERK antibody (Cell Signaling Technology antibody 4370, diluted 1:400) overnight at 4 °C in a humidified chamber, followed by SignalStain boost anti-rabbit IgG polymer (Cell Signaling Technologies) and diaminobenzidine (DAKO) as recommended by the manufacturer. Immunostaining for PKCα used anti-PKCα antibody from Abcam (ab32376) as we have described (26 (link)). Controls for staining specificity involved omission of primary antibody or replacement of the primary antibody with normal rabbit serum. Sections were counterstained with hematoxylin and dehydrated before mounting with coverslips.
6
Retinal Cryosectioning and Flat Mounting
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For retinal cryosections, the eyeballs were embedded in optimal cutting temperature compound at −20°C overnight. 8‐μm serial cryosections were prepared. The slices were probed with primary antibodies (1:100) at 4°C overnight followed by the incubation with fluorescence‐labeled secondary antibodies (1:1000, 1 hur) and counterstained with 4′6‐diamidino‐2‐phenylindole (DAPI, 1:1000, 5 min). For retinal flat mounts, eyeballs were fixed in 4% paraformaldehyde for 30 min and the retinas carefully were dissociated. After incubated with primary (1:100, 4°C, overnight) and respective secondary antibodies (1:1000, 1 h). The retinas were washed extensively and flat mounted on the slides. The primary antibodies included CD31 antibody (BD Bioscience, San Jose, CA, 550274), nerve/glial antigen 2 (NG2) antibody (Santa Cruz Biotechnology, Dallas, TX, sc‐166 251), ionized calcium‐binding adapter molecule 1 (Iba1) antibody (Wako Chemicals, Osaka, Japan, 019‐19 741), glial fibrillary acidic protein (GFAP) antibody (Abcam, Cambridge, UK, ab302644), HMGB1 antibody (Santa Cruz, sc‐74 085), β‐tubulin antibody (Abcam, ab215037), protein kinase C‐α antibody (Abcam, ab32376), and microtubule‐associated protein 2 antibody (Abcam, ab254264). The samples were imaged using a confocal microscope (Carl Zeiss LSM700) or an automated upright fluorescence camera (Leica DM4000B, Germany).
7
Immunohistochemical Analysis of Retinal Tissues
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Retinal tissues were cryoprotected in 30% sucrose for 24 h and embedded in OCT medium (Thermo Scientific, Cat# 6502). Ten-micrometer tissue sections were cut at -20°C in a cryostat (Thermo Scientific) and collected on the poly-L-lysine coated slides. After blocking with 5% BSA and 0.1% Triton X-100 in PBS, the retinal sections were incubated overnight at 4°C with the following primary antibodies: GFAP (1:200, Abcam Cat# ab68428, RRID: AB_1209224), NeuN (1:300, Abcam Cat# ab177487, RRID: AB_2532109), Calretinin (1:500, Santa Cruz Biotechnology Cat# sc-365956, RRID: AB_10846469), Calbindin (1:200, Santa Cruz Biotechnology Cat# sc-365360, RRID: AB_10841576), Rhodopsin (1:400, Abcam Cat# ab5417, RRID: AB_304874) and PKCα (1:400, Abcam Cat# ab32376, RRID: AB_777294). The retinal sections were washed and incubated for 3 h at room temperature with the fluorophore-conjugated secondary antibodies. The retinal sections were observed using an Olympus IX-73 microscopy and the fluorescent signals were analyzed by Image J.
8
Western Blot Analysis of Signaling Proteins
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Protein samples were extracted as mentioned previously. Following SDS–polyacrylamide gel electrophoresis, western blotting was performed by standard procedure. An equal amount (40 μg) of each sample was loaded. Antibodies against STLK3 (dilution 1:1,000, #ab128894), MST1 (dilution 1:1,000, #ab245190), p-PKCα (dilution 1:1,000, #ab76016), and PKCα (dilution 1:1,000, #ab32376) were purchased from Abcam (Cambridge, MA). Antibody against β-actin (dilution 1:10,000, #66009-1-Ig) was purchased from Proteintech (Wuhan, China).
9
Immunohistochemical Analysis of Xenografts
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Immunohistochemistry (IHC) was performed for the xenografts extracted for ICA-1 T treatments. For each sample, blocking was performed following deparaffinization of the sections, and citrate microwave antigen retrieval. PKC-α was detected after incubated with rabbit monoclonal anti-PKC-α (1:100 dilution; ab32376, Abcam) for 60 minutes. ‘EnVision’ detection system was used. Separate sections were subsequently incubated over night with purified rabbit polyclonal anti-PKC-ι (1:100 dilution; ab5282, Abcam), rabbit polyclonal anti-PKC-ζ (1:100 dilution; ab59364, Abcam) and rabbit monoclonal anti-Vimentin (5741S, Cell Signaling Technology). This research was assisted in part by the Analytic Microscopy Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI designated Comprehensive Cancer Center (P30-CA076292).
10
Quantitative Protein Expression Analysis
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Total protein was harvested from cells. Protein concentrations were determined by a BCA kit. An equal amount of protein was isolated with a 12% SDS-PAGE. The isolated protein was transferred onto PVDF membranes. After sealed with non-fat milk, the membranes were incubated with primary antibodies, such as anti-PDSS2 (ab251797, 1: 1000, Abcam, Cambridge, MA), anti-Nrf2 (ab137550, 1: 1000, Abcam), anti-PKCα (ab32376, 1: 1000, Abcam), anti-p53 (ab32389, 1: 1000, Abcam), anti-FSP1 (ferroptosis-suppressor-protein 1, ab197896, 1: 1000, Abcam), and anti-GAPDH (ab9485, 1:2500, Abcam) at room temperature for 2 hours. Then the membranes were incubated with secondary goat antirabbit antibody (ab6721, 1: 2000, Abcam). Finally, the bands were visualized with an enhanced chemiluminescent kit (Pierce) and analyzed with quantified LAS3000 imaging system (Fujifilm Corporation, Japan).
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