KCa3.1 inhibitor TRAM-34, NF-κB inhibitor PDTC and STAT3 inhibitor AG490 were purchased from MCE (Shanghai, China). All these inhibitors dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Antibodies against KCa3.1 (60276-1-Ig) and Interleukin (IL)-1β (16806-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies for inducible nitric oxide synthase (iNOS) (ab178945), matrix metalloproteinase (MMP)-9 (ab228402), vascular cell adhesion molecule (VCAM)-1 (ab134047), STAT3 (ab68153), phospho (p)-STAT3 (ab76315), horseradish peroxidase (HRP)-conjugated IgG (ab205718, ab205719) were purchased from Abcam (Cambridge, MA, USA). Antibodies against β-tubulin (15115S), NF-κB p65 (8242T), p-NF-κB p65 (3033T), p38 MAPK (8690T) and p-p38 MAPK (4511T) were purchased from CST (Danvers, MA, USA). Antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (BL006B) and β-actin (BL005B) were purchased from Biosharp (Hefei, China). Primary antibody for Histone H3 (EM30605) was purchased from HUABIO (Hangzhou, China)
Ab205718 ab205719
Manufactured by Abcam
Sourced in United States
Ab205718/ab205719 are recombinant antibodies. They are designed for use in immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab68153, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
Bl006b, by Biosharp (1 mentions)
Bl005b, by Biosharp (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ab76315, by Abcam (1 mentions)
Ab228402, by Abcam (1 mentions)
Sc79 s7863, by Selleck Chemicals (1 mentions)
Mhy1485 s7811, by Selleck Chemicals (1 mentions)
Cisplatin, by Qilu Pharmaceutical (1 mentions)
Ab33911, by Abcam (1 mentions)
Ab183218, by Abcam (1 mentions)
Ab216995, by Abcam (1 mentions)
Ab106556, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
7 protocols using ab205718 ab205719
1
Therapeutic Inhibitors Targeting Inflammation
2
Investigating Multidrug Resistance Mechanisms
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MSA (541,281) and dansylcadaverine (30,432) were purchased from Sigma-Aldrich (St. Louis, United States). Cisplatin was purchased from Qilu Pharmaceutical (Jinan, China). CQ (MB1668) and 3-MA (MB5063) were purchased from meilunbio (Dalian, China). SC79 (s7863) and MHY1485 (s7811) were purchased from Selleck (shanghai, China). The following antibodies were used, MDR1 (22336-1-AP), ALDH1A1 (60171-1-Ig), Bcl-2 (12789-1-AP), Bax (50,599-2-lg), Caspase-3 (19677-1-AP), Parkin (14060-1-AP), P62 (66,184-1-lg), LC3 (14600-1-AP), Akt (10176-2-AP), phospho-Akt (ser473) (66,444-1-lg), phospho-Akt (thr308) (29163-1-AP), mTOR (66,888-1-lg) were purchased from Proteintech (Wuhan, China). phospho-mTOR (ser2448) (#5536), GAPDH (#5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies (ab205718, ab205719) were procured from Abcam (Cambridge, United Kingdom).
3
Protein Expression Analysis in Rheumatoid Arthritis
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RASFs (8 × 105) and serum samples were exposed to RIPA buffer to extract total proteins. Protein concentration was quantified as per the instruction of a bicinchoninic acid assay (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Afterward, the protein samples were loaded onto 10% separating gel and then shifted onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA) (ab29; 1:1000; Abcam, Cambridge, MA, USA), Cyclin D1 (ab16663; 1:200; Abcam), Cyclin E1 (ab33911; 1:1000; Abcam), BCL2-associated x protein (Bax) (ab32503; 1:5000; Abcam), B-cell lymphoma-2 (Bcl2) (ab32124; 1:1000; Abcam), IL-1β (ab216995; 1:1000; Abcam), TNF-α (ab183218; 1:1000; Abcam), RSPO1 (ab106556; 1:500; Abcam), and β-actin (ab8226, 1:1000; Abcam). Then, the membrane was incubated with HRP-conjugated secondary antibody (ab205718/ab205719; 1:5000; Abcam), and the protein bands were visualized by electrochemiluminescence. The intensities of protein bands were quantified using Image Lab analysis software (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Oxidative Stress and Inflammation
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Cells were firstly lysed in RIPA buffer (Sigma-Aldrich, USA) and protein concentrations of each homogenate were measured using a commercial BCA protein assay Kit (Thermo Fisher Scientific, USA). 30 μg of proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk in TBST, and stained overnight at 4 °C with primary antibodies against Nrf2 (1:1000, ab137550, Abcam), HO-1 (1:2000, ab13243, Abcam), eNOS (1:5000, ab76198, Abcam), NQO1 (1:10,000, ab80588, Abcam), caspase-1 (1:1000, ab238979, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), HMGB1 (1:10,000, ab79823, Abcam), or β-actin (1:5000, ab115777, Abcam). Samples were then stained with goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:10,000, ab205718/ab205719, Abcam) at room temperature for 2 h. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific, USA). The proteins were quantified using Quantity One software (Bio-Rad, USA).
5
Western Blot Analysis of Apoptosis Markers
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Cells were lysed using RIPA lysis buffer (Beyotime) on ice and centrifuged (20 min, 14, 000g, 4°C) to extract total protein. Thereafter, the protein samples were denatured by heating at 100°C for 3–5 min. After measuring protein concentration with BCA protein assay kit (Abcam, Cambridge, UK), extracted protein samples (about 30 μg/lane) were separated using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Sangon Biotech), and protein was then transferred to nitrocellulose membranes (Invitrogen). After blocking with 5% skim milk (Beyotime), the membranes were then incubated with the primary antibodies at 4°C for 12–16 h: B-cell lymphoma-2 (Bcl-2; ab194583, 1:500, Abcam), BCL2-associated X protein (Bax; ab77566, 1:1000, Abcam), SOX4 (ab70598, 1:500, Abcam), β-actin (ab227387, 1:5000, Abcam). Afterwards, these membranes were probed with corresponding secondary antibody (ab205718/ab205719, 1:4000, Abcam). Finally, the combined signals were detected by enhanced chemiluminescence reagent (Solarbio, Beijing, China). Quantification of protein levels was determined using ImageJ software. The protein abundance was normalized by β-actin. All experiments were repeated three times.
6
Characterizing VAV1 and CELF2 Expression in RN-Sc Cells and Spinal Cord Tissues
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Western blot analysis was performed as previously described37 (link),38 (link). Proteins were extracted from RN-Sc cells and spinal cord tissues using ice-cold RIPA Lysis Buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Thermo Scientific) and quantified using the Bio-Rad Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Next, proteins (30 μg/sample) were subjected to 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocked with 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against VAV1 (#2502, 1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA), CELF2 (ab186430, 1:2000 dilution, Abcam, Cambridge, UK), and β-actin (ab6276, 1:5000 dilution, Abcam). Next, the membranes were incubated for 1 h at room temperature with goat-anti-rabbit/mouse secondary antibody conjugated with horseradish peroxidase (ab205718/ab205719, 1:5000 dilution, Abcam). Finally, proteins bands were visualized through SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).
7
Protein Expression Analysis in Cells
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Cytoplasmic and nuclear proteins were extracted with an NEPER Nuclear and Cytoplasmic Extract Kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Aliquots of cell lysates were separated using a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electroblotted onto nitrocellulose membranes (Bio-Rad), as previously described (Xie et al., 2014 (link)). The membranes were incubated with antibodies raised against ENO1 (#ab155102) and β-catenin (#ab32572) (both from Abcam, San Francisco, CA, USA), and those recognizing β-actin (#4970), N-cadherin (#13116), E-cadherin (#3195), snail family transcriptional repressor 2 (SLUG) (#9585), Vimentin (#5741), MMP-2 (#40994), MMP-9 (#13667), lamin A (#86846), and snail family transcriptional repressor (SNAIL) (#3879) (all from Cell Signaling Technology, Beverly, MA, USA) overnight, followed by the addition of horseradish peroxidase-linked anti-rabbit/mouse immunoglobulin G (IgG) (Abcam #ab205718/ab205719) and enhanced chemiluminescence visualization of the immunoreactive protein bands.
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