Following the desired treatments, CHO cells were washed and harvested on ice, and then lyzed with ice-cold protein extraction buffer (Beyotime, Nanjing, China). The protein concentration was determined using BCA protein assay kit (ComWin, Beijing, China). Equal amounts of protein were loaded into gel wells, separated by electrophoresis on SDS-polyacrylamide gels and transferred onto PVDF transfer membrane (Millipore, Bedford, MA). The transferred blots were blocked with blocking agents (5% w/v BSA and 0.05% Tween-20 in TBS) for 1 h at room temperature. Blots were then incubated in a certain proportion of specific primary antibodies: anti-GRP78 (ab25192 Rabbit monoclonal, Abcam), anti-ATF-6 (ab203119 Rabbit polyclonal, Abcam), anti-PERK (C33E10 Rabbit polyclonal, Abcam), anti-IRE1 (ab37073 Rabbit polyclonal, Abcam), anti-CHOP (ab11419 Mouse monoclonal, Abcam), anti-Beclin1 (ab55878 Rabbit polyclonal, Abcam), anti-LC3B (ab81785 Rabbit polyclonal, Abcam) (1:1000) overnight at 4°C. These membranes were rinsed 3 times for 10 min each with TBS-Tween (Sigma, St. Louis, MO, USA) and incubated with HRP conjugated secondary antibodies (1:5000) (CWBio, Beijing, China). After washing, protein bands were visualized by an enhanced chemiluminescence detection kit (ComWin, Beijing, China). Each band density was quantified using Image J software (GE Healthcare, Piscataway, New Jersey, USA).
Ab55878
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab55878 is a primary antibody product from Abcam. It is a polyclonal antibody that targets a specific protein. The core function of this product is to enable detection and analysis of the target protein in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab48394, by Abcam (5 mentions)
Ab56416, by Abcam (3 mentions)
Ab81785, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab3280, by Abcam (2 mentions)
Ab91526, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab128025, by Abcam (2 mentions)
Pvdf transfer membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by CWBIO (1 mentions)
Imagej software, by GE Healthcare (1 mentions)
Protein extraction buffer, by Beyotime (1 mentions)
Ab11419, by Abcam (1 mentions)
Ab203119, by Abcam (1 mentions)
Ab37073, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab63817, by Abcam (1 mentions)
Ab61392, by Proteintech (1 mentions)
Ab76310, by Abcam (1 mentions)
14 protocols using ab55878
1
Revealing ER Stress and Autophagy Markers
2
Protein Extraction and Western Blot Analysis
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Total cellular proteins were extracted with cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA) and measured by BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were resolved by 10% Tris-glycine SDS polyacrylamide gels and electrotransferred to PVDF membranes (Millipore, Billerica, MA). Western blot analysis was performed as previously described19 (link). The following antibodies were used (company, catalogue number, and dilution): anti-N-cadherin (Santa Cruz, sc-7939, 1:500), anti-3-NT (Abcam, ab61392, 1:500), anti-NOX4 (Proteintech, 14347-1-AP, 1:500), anti-DUSP6 (Abcam, ab76310, 1:500), anti-NRF2 (Abcam, ab31163, 1:500), anti-GSTP (Santa Cruz, sc-134469, 1:500), anti-SQSTM1/P62 (Abcam, ab56416, 1:800), anti-Beclin1 (Abcam, ab55878, 1:1200) and anti-LC3B (Abcam, ab63817, 1:1000).
3
Protein Extraction and Western Blot Analysis
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The tissue samples were homogenized in sodium dodecyl sulphate (SDS) buffer containing the protease inhibitor PMSF. The homogenates were incubated on ice for 20 min and then centrifuged at 12000 rpm for 30 min at 4 °C. The supernatant was collected and equal volume of 2 × SDS buffer was added. The mixture was boiled for 10 min and preserved at -20 °C. The protein extracts (50 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (Millipore, United States). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 90 min and then incubated with primary antibodies against Beclin-1 (3 μg/mL; ab55878, Abcam, Cambridge, United Kingdom), LC3 (2 μg/mL; ab48394, Abcam), 4E-BP1 (2 μg/mL; ab2606, Abcam), and actin (0.5 μg/mL, ab3280, Abcam). The protein bands were detected with secondary antibodies conjugated to horseradish peroxidase (1:5000, Abcam, United Kingdom) and visualized with enhanced chemiluminescence reagents. Each band was quantified through densitometry, and the results are presented as the relative expression of each protein from different samples.
4
Autophagy Markers in Neurodegeneration
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Immunofluorescence was used to evaluate the distribution and expression of LC3-II, Beclin1, and P62 in SH-SY5Y cells from normal control and OGD/R groups. For this purpose, the cells were incubated with antibodies against LC3 (1:800; Novus; NB600-1384), Beclin1 (1:100; Abcam Cat# ab55878) and P62 (1:100; Abcam Cat# ab91526), respectively, in a humidified container at 4°C for 12 hr. The cells were rinsed in PBS for 3 times, and incubated with TRITC conjugated anti-rabbit IgG (1:100, Proteintech) at room temperature for 4 hr. 4, 6-diamidino-2-phenylindole (DAPI, 0.0001%, Sigma) was applied to stain nuclei. The cells were examined by a laser confocal microscope (Nikon D-Eclipse C1, Japan).
5
Immunohistochemical Analysis of Protein Expression
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Immunohistochemistry was performed on the sections of the TMA blocks or of tumors developed in the mouse model. The Ultraview Universal DAB detection kit (Ventana, Ventana medical system, Tuscon, AR) was used. Antibodies against phospho-AKT (1/100, S473-r, Santa Cruz biotechnology, CA), phospho-mTOR (1/100, 49 F9, Cell Signaling, CA), LC3B (1/1000, ab51520 abcam, Cambridge UK) or Beclin 1 (1/250, ab55878 abcam) were applied for 30 min. DAB was used as a chromogen and hematoxylin as a counterstain. Normal mouse or rabbit IgG at the same concentration as the primary antibody were used as negative control and synaptophysin (1/100, Polyclonal, SP11, Thermofisher Scientific) as positive control (Additional file 1 : Figure S1). Two investigators blinded for clinical data independently evaluated immunostaining in samples containing more than 100 NB cells. Immunostaining scores were established by a semi-quantitative optical analysis assessing the percentage of positive cells in each sample: 0 = all cells negative, 1 + = 1 to 25%, 2 + = 26 to 50%, 3 + = 51 to 75% and 4+ more than 75% of positive tumoral cells.
6
Autophagy Protein Expression Analysis
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Western blot analysis was performed to measure the expression of beclin-1 and LC3-II/I, proteins associated with autophagy (25 (link)). HK-2 cells were lysed using RIPA lysis buffer (Teknova, Inc.) at 4°C for 10–15 min. Protein concentration was determined using bicinchoninic acid assay method. Equal amounts of 50 µg protein extract were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk diluted in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 90 min. The membranes were then immunoblotted for Beclin1 (1:1,000; cat. no. ab55878; Abcam), LC3 (1:1,000; cat. no. ab48394; Abcam), and β-actin (1:1,000; cat. no. ab3280; Abcam) at 20–27°C for 2 h before incubation with secondary antibodies conjugated to horseradish peroxidase-conjugated goat anti-rabbit Immunoglobulin G (1:5,000, cat. no. ab7074; Abcam) at 37°C for 20 min and visualized with enhanced chemiluminescence reagent (Vazyme). Immunoreactive bands were analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).
7
Immunofluorescence Analysis of Autophagy Markers
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Antibodies against Beclin 1 (ab55878), LC3B (ab192890), NMMHC IIA (ab75590), F-actin (ab205), Atg9A (ab108338), MAP2 (ab11267), TGN46 (ab2809), NeuN (ab104224), and cytochalasin D (ab143484) were obtained from Abcam (Cambridge, UK). L-glutamine (25030–081), neurobasal medium (21103049), B27 supplement (50×, minus antioxidants, 10889–038), soybean trypsin inhibitor (17075029), Alexa Fluor 594 goat anti-mouse antibody (A11032) and Alexa Fluor 488 donkey anti-rabbit antibody (A21206) were obtained from Thermo Fisher Scientific (San Jose, CA, USA). Cytosine arabinoside (C6645), poly-L-lysine (P1274), D-( + )-glucose (G7021), and 3-MA (M9281) were from Sigma-Aldrich (St. Louis, MO, USA). Blebbistatin (S7099) was obtained from Selleck Chemicals (Houston, TX, USA). ExFect Transfection Reagent (T101–01) was purchased from Vazyme Biotech Co., Ltd (Nanjing, China).
8
Western Blot Analysis of Autophagy Markers
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Cells (1×106) were washed twice with PBS and incubated with pyrolysed solution containing 0.1 M phenylmethylsulfonyl fluoride. Cell lysates were centrifuged at 13,000 × g for 20 min at 4°C. Proteins were quantified by Bradford assay and 10 µg were separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Following blocking with 5%non-fat dry milk in TBS for 1 h at room temperature, membranes were incubated with rabbit anti-Beclin 1 (1:500 dilution, ab55878), anti-GAPDH antibody (1:1,000 dilution, ab9485) or rabbit anti-microtubule-associated protein 1A/1B-light chain 3 (LC3)B (1:1,000 dilution, ab81785) (all from Abcam, Cambridge, UK) primary antibodies at 4°C overnight. Following fives washes with TBS with 0.5% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000 dilution, ab6717; Abcam) for 30 min at room temperature. Specific bands were observed using enhanced chemiluminescene (ECL; Thermo Fisher Scientific, Inc.) and detected using a Bio-Rad ChemiDoc XRS image system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
9
Quantitative Analysis of Autophagy Markers
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Total protein was extracted using a kit (KGP250; Nanjing Keygen Biotech Co. Ltd., Nanjing, China). Whole cell lysat was separated by 10-15% SDS-PAGE then transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (0.1% Tween 20 in TBS) for 1 hr at room temperature and incubated overnight at 4°C with antibodies against LC3II (1:500; Abcam, ab62721), Beclin1(1:1000; Abcam, ab55878), P62 (1:1000; Abcam, ab91526), DUSP5 (1:400; Abcam, Cat# ab54939 ), ERK1/2 (1:700; CST, Cat# 4695), p-ERK1/2 (1:1000; CST, Cat# 4370) and GAPDH (1:2000; Santa Cruz Biotechnology; Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:3000; Proteintech Group, Inc., Hubei, China). Immunoreactive bands were visualized using a chemiluminescence kit (ECL kit; Santa Cruz Biotechnology, USA), and protein bands were scanned using Chemi Imager 5500 V2.03 software. The integrated density value (IDV) for each band was calculated with a computer-aided image analysis system (Fluor Chen 2.0). The IDV of LC3II was normalized with the IDV of LC3I, while the other proteins were normalized with the IDV of GAPDH.
10
Western Blot Analysis of Cellular Signaling
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Total cell extracts were prepared using ice-cold lysis buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Sigma-Aldrich). Proteins (20 μg/lane) were separated by SDS-polyacrylamide gel electrophoresis (10–15% gels) and transferred onto nitrocellulose membranes (Sigma-Aldrich). Membranes were blocked in 5% non-fat milk in TBS/0.1% Tween 20 for 2 h prior to immunoblotting overnight with antibodies against LC3B (1:500, ab48394; Abcam), phospho (p)-AMPKα (1:500; #2535, Cell Signaling Technology, Inc., Shanghai, China), p-mTOR (1:1000; #2971, Cell Signaling Technology), p-NF-κB (p65) (1:500; sc-166,748, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), TGFβ1 (1:1000; ab31013, Abcam), p-JAK2 (1:1000; ab195055, Abcam), p-STAT3 (1:800; ab30647, Abcam), Beclin-1(1:500; ab55878, Abcam) and β-actin (1:1000, Santa Cruz Biotechnologies). Incubation with the secondary fluorescent-labeled antibody (Alexa Fluor 488) was performed for 2 h at room temperature in the dark. The proteins were visualized by enhanced chemiluminescence (Amersham Bio-sciences, NJ, USA).
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