The nasal tissues in AR group and NR group were prepared into a homogenate and centrifuged at 2,000 r/min for 20min, and the supernatant was collected. The protein concentration was measured using BCA kit (Solarbio, Beijing, China), 40μl protein sample and 10% SDS gel buffer were mixed by 1:1 and heated 5min at 95°C for protein denaturation. Then, the mixture was transferred onto a polyvinylidene difluoride (PVDF) membrane at 80V (Merck, Darmstadt, Germany) for 30 min, and then the PVDF membrane was blocked with TBST solution containing 5% defatted milk powder at 4°C for 1h, added with rabbit anti-human FOS (1:1000, ab190289, Abcam), Bcl-2 (1:1000, ab196495, Abcam), Bax (1:1000, ab263897, Abcam), Beclin1 (1:1000, ab62557, Abcam), p62 (1:1000, ab155686, Abcam), and β-actin (1:2000, orb178392, Biorbyt, Cambridge, UK) polyclonal antibodies which were diluted with TBST solution containing 3% FBS protein, and then incubated overnight at 4°C. After re-warming, the PVDF membrane was incubated with horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1:1000, ABIN101988, antibodies-online, Aachen, Germany) for 1h, washed, and developed with ECL luminescent substrate for 3-5min. The protein expression level was normalized using β-actin, and the gray scan and quantification were performed with Image J(NIH) software.
Ab155686
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab155686 is a laboratory equipment product manufactured by Abcam. The core function of this product is to facilitate specific experimental procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab48394, by Abcam (3 mentions)
Ab205718, by Abcam (2 mentions)
Ab51520, by Abcam (2 mentions)
Ripa lysis solution, by Beyotime (2 mentions)
Ja31 14, by Cell Signaling Technology (2 mentions)
Maplc3 2, by Cell Signaling Technology (2 mentions)
Protein a g, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab190289, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab263897, by Abcam (1 mentions)
Orb178392, by Biorbyt (1 mentions)
Ab62557, by Abcam (1 mentions)
Abin101988, by Antibodies-online (1 mentions)
Bca kit, by Solarbio (1 mentions)
Ab101318, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bca detecting kit, by Beyotime (1 mentions)
14 protocols using ab155686
1
Western Blot Analysis of Nasal Tissues
2
Protein Isolation and Western Blot Analysis in CRC Cells
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The proteins were isolated from CRC cells using RIPA lysis solution (Beyotime, Shanghai, China). The proteins were quantified through using a BCA detecting kit (Beyotime), and then the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. After incubating with 5% non-fat milk for 1 h, the PVDF membrane was incubated with specific antibodies overnight. The antibodies against Bcl-2 associated X, apoptosis regulator (Bax; ab32503), Caspase-3 (ab13847), light chain 3 I/II (LC3I/II; ab51520), Beclin-1 (ab210498), p62 (ab155686), ZNF281 (ab101318) and β-actin (ab8226) were obtained from Abcam (Cambridge, MA, USA). The membrane was then incubated with a secondary antibody (ab205718, Abcam) for 1 h. The protein images were obtained through the enhanced chemiluminescent (ECL) system (Beyotime).
3
Zinc and Chloroquine Autophagy Regulation
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Cells were treated with 0, 10, or 50 μM of Zn2+ and 10 μM of CQ for 24 h. Then, cells were washed twice with cold PBS and lysed with 35 μL of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM DTT, 10 mM 13-GP, 0.1 mM Na3VO4, and protease inhibitors). Lysates were vortexed for 30 min at 4 °C and centrifuged at 10,000× g to remove aggregates. Lysates were boiled at 95 °C and placed in ice for 1 min. 20 μL of each sample were loaded onto a 12% or 14% polyacrylamide gel. After transfer, membranes were blocked with 5% milk in TBS-Tween 0.1% for 1 h at room temperature. Primary antibodies were diluted in blocking solution—microtubule-associated proteins 1A/1B light chain 3B (LC3) (L8918, Sigma-Aldrich, Darmstadt, Germany) at 1:500, p62 (ab155686, Abcam, Cambridge, UK) at 1:1000, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, Cambridge, UK) at 1:1000—and incubated overnight at 4 °C. Anti-rabbit or anti-mouse horseradish peroxidase (HRP) secondary antibodies (1:1000; GE Healthcare, Chicago, IL, USA) were used.
4
Western Blot Analysis of EMT Markers
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Western blot assays were performed as previously described34 (link). Briefly, the total cellular proteins were extracted by using reagents in a Radio Immunoprecipitation Assay kit (Beyotime, Shanghai, China). The extracted proteins were then separated by SDS-PAGE, and the individual protein bands were transferred onto a PVDF membrane, which was subsequently blocked with 5% skim milk. The membrane was then incubated with primary antibodies against E-cadherin (1:800, ab76055, Abcam, Cambridge, MA, USA), FN (1:5000, ab45688, Abcam, USA), β-catenin (1:8000, ab32572, Abcam, USA), α-SMA (1:1000, ab28052, Abcam, USA), LC3B (1 μg/mL, ab48394, Abcam, USA), p62 (1:2000, ab155686, Abcam, USA), or the internal reference GAPDH (1:20000, ab128915, Abcam, USA). Next the membrane was incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies. An enhanced chemiluminescence detection system was used to detect areas of luminescence, and the relative-staining intensity of each protein band was quantitated using Image Plus Pro software (Ver. 6.0, Media Cybernetics, Inc., Rockville, MD, USA).
5
Western Blot Analysis of Autophagy and GFRA1 in Osteosarcoma
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Osteosarcoma cells were lysed with RIPA lysis solution (Beyotime, Shanghai, China). Proteins extracted from osteosarcoma cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with nonfat milk for 1 h at room temperature and then incubated at 4°C overnight with the following primary antibodies: anti-LC3 (1:5000, ab51520, Abcam), anti-Beclin-1 (1:8000, ab210498, Abcam), anti-p62 (1:5000, ab155686, Abcam), anti-GFRA1 (1:5000, ab84106, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:20000, ab37168, Abcam). Afterward, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:5000, ab205718, Abcam) for 2 h at room temperature. Protein bands were detected through the enhanced chemiluminescence system (Beyotime).
6
Immunofluorescence Assay for p62 Protein
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For the immunofluorescence assay, 4 μm thick sections were then washed with PBS and permeabilized with 0.2% Triton X-100 at room temperature for 15 min. After blocking with 5% bovine serum albumin, slides were incubated with primary antibodies against rabbit anti-p62 (1 : 200; ab155686; Abcam, Cambridge, MA) overnight at 4°C. Subsequently, the sections were stained with Alexa Fluor-594-conjugated goat-anti-rabbit IgG H&L secondary antibody (1 : 1000; ab150088; Abcam, Cambridge, MA) for 1 h at 37°C. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 10 min at 37°C in the dark. Finally, the coverslips were mounted on slides on an antifluorescence quencher, and the images were captured using a fluorescence microscope (DM60008, Leica, Germany) and analyzed with Image-Pro Plus (Medium Cybernetics, Bethesda, MD, USA).
7
Western Blot Analysis of Cellular Signaling
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The antibodies specific against JUN (9165S), JUND (5000S), P-JUN Ser63 (2361S), P-JUN Ser73 (9164S), AKT (9272S), p-AKT Ser473 (9271S), RELA (8242S), PIK3CA (4255S), PARP (9542S), GAPDH (5174S), NFE2L2 (12721S), MTOR Pathway Antibody Sampler Kit (9964S), Autophagy Antibody Sampler Kit (4445S), RPS6KB1 Substrates Antibody Sampler Kit (2903S) and ULK1 Antibody Sampler Kit (8359T) were purchased from Cell Signaling Technology. Antibodies to GFP (sc-390394), ETS1 (sc-55581), SP1 (sc-14027) and MYC (sc-788) were bought from Santa Cruz Biotechnology. Antibodies that are specific against NFKB1 (ab32360) and SQSTM1 (ab155686) were bought from Abcam. Antibodies against ACTB (A1978) and TUBA/α-tubulin (T6199) were bought from Sigma Aldrich Corporation. Western blotting was performed as described in our previous publication.73,74 (link)
8
Immunoblotting and Immunoprecipitation Protocol
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Cells were harvested and lysed in WIP lysis buffer (Beyotime, Shanghai, China) for immunoblots and immunoprecipitation. Protein concentrations of the lysates were determined by the BCA protein assay system (Beyotime). Equal amounts of protein were separated by 10% SDS-PAGE, transferred to PVDF membrane (Millipore, Billerica, MA). The used antibodies were anti-LC3B (No. 3868, Cell Signaling Technology, 1 : 1000); anti-ATG5 (10181-2-AP, Proteintech, 1 : 1000); anti-ATG7 (ab133528, Abcam, 1 : 50,000); anti-p62 (ab56416, Abcam, 1 : 2000); anti-p62 (ab155686, Abcam, 1 : 2000); anti-BECN1 (No. 3495, Cell Signaling Technology, 1 : 1000); anti-NRF2 (ab137550, Abcam, 1 : 1000); anti-GATA4 (ab12465, Abcam, 1 : 1000); anti-p65 (No. 8242, Cell Signaling Technology, 1 : 2000); anti-p-p65 (No. 3033, Cell Signaling Technology, 1 : 2000); anti-GAPDH (sc-365062, Santa Cruz, 1 : 1000); anti-ICAM-1 (sc-8439, Santa Cruz, 1 : 1000); anti-eIF2α (No. 5324, Cell Signaling Technology, 1 : 1000); anti-p-eIF2α (No. 3398, Cell Signaling Technology, 1 : 1000); anti-Chop (No. 5554, Cell Signaling Technology, 1 : 1000); and anti-ATF4 (10835-1-AP, Proteintech, 1 : 1000). Protein A/G was purchased from Thermo Fisher, rab-IgG from Beyotime, horseradish peroxidase secondary antibody from Amersham Pharmacia Biotech, and ECL kit from Thermo Fisher.
9
Analyzing Neuronal Survival and Autophagy
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A subset of sliced tissues was used for immunohistochemical labeling with antibody against the neuronal nuclei (NeuN) or microtubule-associated protein 2 (MAP2) to assess the surviving neuronal cells in the brain. To detect autophagy activation and maturation, a subset of slides were immunolabeled with antibody against LC3 (ab48394, autophagosome marker) and antibody against SQSTM1/P62 (ab155686), purchased from Abcam. Briefly, slides were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in 0.1% Triton X-100 with 1% milk/1% goat serum, and immunolabeled with primary antibodies. Immunoreactivity was visualized with secondary antibodies from Molecular Probes (Carlsbad, CA, USA). Cells were mounted with Prolong Gold antifade reagent with DAPI to label cell nuclei (Thermo Fisher). To investigate whether EV-TPP1 treatments affected protein aggregates in the brain of CLN2 mice, primary antibodies to subunit c of mitochondrial ATP synthase (ab181243, 1:500 dilution) were used for the accumulation of lysosomal storage bodies and secondary antibody goat anti-rabbit IgG H+L Alexa Flour 488 (ab11008).
10
Immunohistochemical Analysis of Autophagy Markers
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Immunohistochemical analysis of p62, LC3, and Beclin1 markers was performed on TMA sections with Autostainer Link 48 (Agilent, Glostrup, Denmark). EnVision FLEX epitope unmasking solution, pH 6.1 (K8005, Agilent), was used for LC3B antibody epitope unmasking. EnVision FLEX epitope unmasking solution, pH 9.0 (K8004, Agilent), was used for p62 and Beclin1 antibody epitope unmasking. Immunohistochemical analysis was performed with the visualization system EnVision FLEX (Agilent). The following primary antibodies were used: polyclonal rabbit anti-human p62 antibody (ab155686, Abcam, Cambridge, UK) at 1:500 dilution; monoclonal recombinant rabbit anti-human Beclin-1 antibody (EPR20473, Abcam) at 1:50 dilution; and polyclonal rabbit anti-human LC3B antibody (ab4839zhen, Abcam) at 1:200 dilution. The percentage of positive cells was recorded. The microscope magnification was x400. For immunohistochemical interpretation, we used the following score: negative (0) expression – without positive cells or with a single positive cell (<1%); low (1+) expression – less than 10% positive cells; moderate (2+) expression – 10%-50% positive cells; and strong (3+) expression – more than 50% positive cells (18 (link)).
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