The Institute of Cell Biology of the Chinese Academy of Sciences acquired the human NB cell lines SK-N-SH and SH-SY5Y. All cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, U.S.A.), supplemented with 10% FBS (Gibco, U.S.A.), 100 units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% carbon dioxide at 37°C. The medium was changed once every 2 days. Rapamycin powder was purchased from Sigma Chemical Co. (U.S.A.). All reagents were diluted in DMSO (Sigma, U.S.A.) and kept at −20°C. Antibodies included anti-Beclin-1 (ab114071, Abcam, U.K.), anti-LC3-I/II (ab114071, Abcam, U.K.), anti-P62 (ab91526, Abcam, U.K.), anti-mTOR (ab109268, Abcam, U.K.), anti-p-mTOR (ab32028, Abcam, U.K.), anti-GAPDH (ab8245, Abcam, U.K.), and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, CA).
Ab114071
Manufactured by Abcam
Sourced in United Kingdom
Ab114071 is a rabbit monoclonal antibody that recognizes PRSS1. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab51520, by Abcam (3 mentions)
Ab91526, by Abcam (2 mentions)
Ab109268, by Abcam (2 mentions)
β actin a5441, by Merck Group (2 mentions)
Catalase, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Calcium oxalate, by Merck Group (1 mentions)
Mouse anti becn1, by Cell Signaling Technology (1 mentions)
Mouse anti becn1, by Abcam (1 mentions)
Rabbit anti lc3b, by Merck Group (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Zb 2301, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Zb 2305, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Af886, by R&D Systems (1 mentions)
Ab170941, by Abcam (1 mentions)
9 protocols using ab114071
1
Modulation of Autophagy in NB Cells
2
Autophagy Regulation via Calcium Oxalate
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Calcium oxalate (Sigma, 455997), 3-methyladenine (Sigma, M9281), rapamycin (Sigma, R0395), 4′,6-diamidino-2-phenylindole (Sigma, D8417), N-acetyl-L-cysteine (Sigma, A7250), catalase (Millipore, 219261-100KU), 2′,7′-dichlorofluorescin diacetate (Sigma, D6883), Lipofectamine 3000 (Invitrogen, L3000008), and rabbit anti-LC3B (Sigma, L7543) for western blot (WB) (1:2000), mouse anti-BECN1 (Cell Signaling Technology, 3495) for WB (1:1000). rabbit anti-LC3B (Abcam, ab51520) (1:1000) and mouse anti-BECN1 (Abcam, ab114071) were used for immunohistochemistry (IHC) (1:500). Mouse monoclonal anti-GAPDH (Proteintech, 60004-1-Ig) was used for WB (1:5000). Mouse and rabbit HRP-conjugated antibodies were obtained from Zhongshan Golden Bridge Biotechnology (ZB-2305, ZB- 2301).
3
Western Blot Analysis of Autophagy Markers
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Whole cell lysates were further processed by SDS-Page followed by Western blotting, as previously described [18 (link)]. Immunodetection with primary antibodies against Map1LC3B (ab51520), Atg12 (ab56465) Sqstm1 (ab96706), Beclin1 (ab114071) (AbCam), UVRAG (U7508) and b-actin (A5441) (Sigma-Aldrich) was performed. Bound secondary HRP-conjugated anti-mouse (A9917) and anti-rabbit (A0545) antibodies (Sigma-Aldrich) were detected by incubating the immunoblots with SuperSignal West Pico Chemiluminescent Substrate (#34077, Pierce, Thermo Fisher Scientific). The luminescent reactivity was then measured using Fusion image capture and further quantified with Bio1D analysis system (PEQLAB Biotechnologie GmbH). Anti-b-actin was used to control equal loading and protein quality.
4
Protein Expression Analysis by Western Blotting
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Whole cell lysates were further processed by SDS-Page followed by Western blotting, as previously described [15 (link)]. Immunodetection with primary antibodies against human Survivin (AF886, R&D Systems, Wiesbaden-Nordenstadt, Germany), Bcl-2 (610539, BD Transduction Laboratories) Beclin1 (ab114071), Map1lc3b (ab51520) and Sqstm1 (ab96706) (AbCam), DRAM1 (PRS4035 Sigma-Aldrich Saint Louis MO USA), ATF4 (ab50546, AbCam) and β-actin (A5441) (Sigma-Aldrich) was performed. Bound secondary HRP-conjugated anti-mouse (A9917) and anti-rabbit (A0545) antibodies (Sigma-Aldrich) were detected by incubating the immunoblots with Super Signal West Pico Chemiluminescent Substrate (#34077, Pierce, Thermo Fisher Scientific, Darmstadt Germany). The luminescent reactivity was then measured using Fusion image capture and further quantified with Bio1D analysis system (PEQLAB Biotechnologie GmbH). The blotted nitrocellulose membranes (Amersham Protran Premium 0.2 μm NC Cat. N. 10600009 Ge Healthcare Life Science, Freiburg Germany) were up to four times stripped with Stripping Buffer (Restore Plus Western Blot Stripping Buffer Cat. N. 46430 Thermo Scientific) as suggested by the company and reprobed in order to detect other proteins. At last, anti-β-actin was used to control equal loading and protein quality.
5
Comprehensive Western Blot Analysis
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Western blot analysis was performed as previously described.22 (link) Cells were lysed in hot 1× sodium dodecyl sulfate sample buffer, and 30 µg samples were loaded into each lane of 4%–12% polyacrylamide gels. The proteins were separated by electrophoresis, and the proteins in the gels were transferred onto nitrocellulose membranes (Pall; Port Washington, NY, USA). The membrane was blocked with 5% nonfat milk for 1 hour at room temperature and then incubated with antibodies against PR (3176; Cell Signaling Technology, Danvers, MA, USA), pAKT (Ser473; 4058; Cell Signaling Technology), AKT (4685; Cell Signaling Technology), phosphor-mTOR (Ser2448; 5536; Cell Signaling Technology), mTOR (2972; Cell Signaling Technology), LC3B (2775; Cell Signaling Technology), ATG3 (3415; Cell Signaling Technology), ATG5 (8540; Cell Signaling Technology), KRAS (ab137739; Abcam, Cambridge, UK); PTEN (ab170941; Abcam), EIG121 (ab156275; Abcam), P62 (ab91526; Abcam), Beclin-1 (ab114071; Abcam), and β-actin (sc-47778; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 16 hours at 4°C. Then, the specific horseradish peroxidase-conjugated rabbit anti-mouse or rabbit anti-mouse IgG was added to the membrane and incubated for 1 hour at room temperature. Detection by the chemiluminescence reaction was carried out using the enhanced chemiluminescence (ECL) kit (Pierce; Rockford, IL, USA).
6
Protein Expression Analysis Workflow
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Total protein content was extracted from the tissues and cells using Protein lysis buffer (C0481, Sigma, St Louis, MO). Following 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane. The membranes were subsequently incubated with the following antibodies to HK2 (dilution ratio of 1 : 1000, ab104836, Abcam), mTOR (dilution ratio of 1 : 2000, ab2732, Abcam), p-mTOR (dilution ratio of 1:1000, ab109268, Abcam), Beclin1 (dilution ratio of 1 : 500, ab114071, Abcam), Bax (dilution ratio of 1 : 1000, ab77566, Abcam), Bcl-2 (dilution ratio of 1 : 1000, ab692, Abcam), 4EBP1 (dilution ratio of 1 : 1000, ab32130, Abcam), p-4EBP1 (dilution ratio of 1:1000, ab47365, Abcam), LC3A/B (dilution ratio of 1 : 1000, ab128025, Abcam), MMP-9 (dilution ratio of 1 : 1000, ab73734, Abcam) and β-actin (dilution ratio of 1:500, Beijing Cwbiotech Co., Ltd., Beijing, China) overnight at 4°C. Afterwards, the horseradish peroxidase (HRP) -labeled goat anti-mouse or anti-rabbit IgG (SPA131 or SA27, dilution ratio of 1 : 500, Solarbio, Beijing, China) was incubated with the membrane at room temperature for 1.5 h. The relative expression of the proteins was measured as previously described 51 (link).
7
Beclin 1 Immunofluorescence by Flow Cytometry
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The fixation and permeabilisation were performed as described above. Cells were resuspended in 100 µL of diluted primary antibody (1:100 in PBS, anti-beclin 1 antibody (2A4) #ab114071, Abcam, Cambridge, UK) and incubated 30 min in the dark at RT. The cells were washed three times by centrifugation in PBS. Then to each probe, 100 µL of diluted fluorochrome-labelled secondary antibody (1:100 in PBS; goat anti-mouse IgG H&L – Alexa Fluor 488 #ab150113, Abcam, Cambridge, UK) was added and incubated 30 min in dark at RT. Then, cells were rinsed with 2.5 mL PBS. In the last step, supernatant was discarded and cells were resuspended in 300 µL PBS and pipetted. The analysis was carried out by flow cytometry.
8
Immunohistochemical Evaluation of Autophagy Markers
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Tissue sections (4 µm thick) for immunohistochemistry were deparaffinized in xylene, followed by rehydration with serially decreased ethanol concentrations. Then, tissues were placed in citrate buffer (pH 6.0) for antigen retrieval (95 ºC, 15 min). Endogenous peroxidase was blocked by 3% H2O2 solution for 10 min at 25 ºC. Sections were incubated with each primary antibody for 2 h at 25 ºC, washed three times with phosphate-buffered saline (PBS), and then incubated with secondary antibodies for 20 min at 25 ºC. Subsequently, sections were incubated with DAB substrate for 5-10 min. Anti-LC3 (ab48394, 1:800) and anti-Beclin1 (ab114071, 1:450) were purchased from Abcam (Cambridge, UK). The expression of autophagy-related proteins was evaluated by measuring the percentage of positively stained cells and the staining intensity. The percentage of positively stained cells was graded as follows: 0, ≤ 5%; 1, 6-35%; 2, 36-65%; and 3, 66-100%. The staining intensity was graded as follows: 0, no staining; 1, buff; 2, yellow; and 3, brown. The final staining score was calculated by multiplying the above-obtained scores. Tumors with an immunoreactive score of 0-3 were designated as negative, whereas those with 4-9 were classified as positive. All sections were submitted to 2 pathologists for evaluation.
9
Monitoring Autophagy Markers in HepG2 and Hep3B
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HepG2 and Hep3B cells were treated for 6 and 24 hours with 100 nM panobinostat. Immunoprecipitation was performed by the use of Pierce® Crosslink Immunoprecipitation Kit (Thermo Scientific, 26147). Beclin1 was precipitated with the antibody ab114071 (AbCam). The final elutes were processed by western blotting to detect Beclin1, UVRAG, Atg12 as described above. Ectopic EGFP-mRFP-MAP1LC3B was detected with a primary antibody for GFP (ab1218) from AbCam.
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