Cells were harvested by centrifugation, washed with PBS and lysed using buffer composed of 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1.0% NP-40, 0.1% SDS, supplemented with protease and phosphatase inhibitors. Protein concentrations were estimated by Bradford assay and equivalent quantities of the lysates were resolved on 4–20% Tris-HCl SDS-PAGE TGX gels (Bio-Rad). Proteins were transferred to nitrocellulose membranes and stained for acetyl-histone H3 (Milipore), acetyl-histone H4 (Milipore), IGF2BP1 (Cell Signaling Technology), IGF2BP3 (IMP-3, Santa Cruz Biotechnology), CD44 (Santa Cruz Biotechnology), CD48 (Abcam), Drosha (Cell Signaling Technology), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology), followed by anti-mouse, or anti-rabbit IgG-HRP (GE Healthcare). Signals were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).
Drosha
Manufactured by Cell Signaling Technology
Sourced in United States
Drosha is a ribonuclease III enzyme that plays a key role in the initial processing of microRNA (miRNA) precursors. It is responsible for cleaving the primary miRNA (pri-miRNA) transcript to produce the precursor miRNA (pre-miRNA) molecule.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Dicer, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Argonaute 2, by Cell Signaling Technology (2 mentions)
Dicer, by Abcam (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Acetyl histone h3, by Merck Group (1 mentions)
Acetyl histone h4, by Merck Group (1 mentions)
Igf2bp1, by Cell Signaling Technology (1 mentions)
Anti mouse or anti rabbit peroxidase, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Genesnap software, by Syngene (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Stim1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
β tubulin, by Merck Group (1 mentions)
C fos, by Merck Group (1 mentions)
11 protocols using drosha
1
Western Blot Analysis of Cell Lysates
2
Renal Protein Profiling by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each sample, 50 µg of renal tissue protein extract was used for electrophoresis on 10% polyacrylamide gels (SDS-PAGE). The immunostaining was carried out with primary antibodies (β-actin/1:1,000, Sigma, USA; IKK-α/1:1,000, Cell Signaling, USA; Argonaute 2/1:1,000, Cell Signaling, USA; Drosha/1:1,000, Cell Signaling, USA; Dicer/1:1,000, Imgenex, India), followed by conjugated secondary antibodies (anti-mouse or anti-rabbit peroxidase/1:5,000, Sigma, USA). Then, the membrane was revealed by chemiluminescence methods using the ECL kit (Millipore, USA), and the images were acquired on GEN-BOX equipment (Syngene, UK) (n = 5). The GeneSnap software and GeneTools (Syngene, UK) were used to identify, analyze, and quantify the gel bands.
3
Western Blot Analysis of RNA-Binding Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in IP lysis buffer as described Srikanth, 2010 #420}. Whole cell lysates were separated on 4–12% Bis-Tris gels (Life Technologies) and proteins were transferred to PVDF membranes (Biorad). Blocking was done using superblock (5% low fat milk, 2% Casein and 1% BSA) or 5% low fat milk Membranes were incubated with the primary antibody in 3% BSA (w/v) in TBST (137 mM NaCl, 20 mM tris, 0.1% Tween-20, pH 7.6) overnight at 4 °C. Membranes were incubated with the HRP-conjugated secondary antibody for one hour at room temperature and bound proteins were visualized using ECL Western blotting detection reagents (GE Healthcare). Blots were imaged using Geliance Gel Imaging system (Perkin Elmer).
Primary antibodies used were STIM1 (4916 S Cell Signaling), Ago2 (2897 S Cell Signaling), β-actin (A1978 from Sigma), Dicer (5362 S Cell Signaling), Drosha (3364 S Cell Signaling), Pumilio1 (12322 S Cell Signaling) and Pumilio2 (12323 S Cell Signaling) Secondary antibodies used were from Jackson ImmunoResearch Laboratories goat anti-rabbit IgG (111-035-044) and goat anti-mouse IgG (115-035-146).
Primary antibodies used were STIM1 (4916 S Cell Signaling), Ago2 (2897 S Cell Signaling), β-actin (A1978 from Sigma), Dicer (5362 S Cell Signaling), Drosha (3364 S Cell Signaling), Pumilio1 (12322 S Cell Signaling) and Pumilio2 (12323 S Cell Signaling) Secondary antibodies used were from Jackson ImmunoResearch Laboratories goat anti-rabbit IgG (111-035-044) and goat anti-mouse IgG (115-035-146).
4
Subcellular Fractionation for Protein Translocation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess translocation of Dicer, Drosha, cJUN and cFOS from cytosol to nucleus, 8 × 105 cells were plated in 10 cm diameter dishes, maintained in 10% FBS for 18 h and then scraped, homogenized on ice in a lysis buffer containing (in mM) 10 HEPES, 1 DTT, 10 KCl, 50 NaF, 0.1 EDTA, 0.1 EGTA, 1 Na3VO4, 0.5 PMSF and 0.1 NP-40 at 4°C, and centrifuged at 1000 g for 10 min to separate the nuclei. The supernatant was centrifuged at 13,200 g for 5 min to yield the cytosolic fraction. The nuclear fraction was lysed in buffer containing (in mM) 20 HEPES, 1 EDTA, 1 EGTA and 0.5 PMSF and analysed for cFOS (Merck Millipore), Dicer (Abcam), Drosha and cJUN (Cell Signaling), all 1:1000. Western blot was performed as described [18 (link)]. Images were digitized with the program CHEMI DOC Quantity One, blots were analyzed in triplicate by densitometry using NIH Image 1.60B5 software, and the results in arbitrary densitometric units (A.D.U.) were normalized for β-actin, β-tubulin or lamin (Sigma Aldrich).
5
Protein Expression Analysis of Key RNA Interference Components
6
Protein Expression Analysis in MV4-11 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extracts from MV4-11 cell lines were harvested using RIPA lysis buffer with protease inhibitors, and protein concentration was determined using a Bio-Rad Protein Assay Dye Reagent kit. SDS-denatured protein was separated via gel electrophoresis and transferred onto a nitrocellulose membrane. Protein was detected via overnight antibody staining with the following antibodies: STAT5A (Santa Cruz L-20), Drosha (Cell Signaling D28B1), Ago2 (Cell Signaling C34C6), p53 (Santa Cruz FL-393), and Actin (Sigma A5441).
7
Protein Lysate Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein lysates were obtained as previously described [18 (link)]. The following antibodies were used: VEGF (Merck Millipore, Darmstadt, Germania), Dicer (Abcam, Cambridge), Drosha (Cell Signalling, Danvers, MA, USA) all 1:1000, mPGES-1 (1:200, Cayman chemicals, Ann Arbor, Mi, USA) or HIF-1α (1:300, BD-Transduction Laboratories, Milan, Italy).
8
Western Blot Analysis of DICER1 and DROSHA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western Blot analyses were performed as previously described [31 (link),34 (link)] with endothelial cells lysed using commercial lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with a premade protease- and phosphatase-cocktail (Thermo Fisher Scientific). Cell debris were removed by centrifugation for 5 min at 15,000× g, 4 °C. SDS-PAGE was performed with equal amounts of protein per lane, followed by transfer to nitrocellulose membrane. Then, membranes were incubated with primary antibody solutions of DICER1 (Cell Signaling Technology; clone D38E7; 1:1000), DROSHA (Cell Signaling Technology; clone D28B1; 1:1000) and β-Actin (clone AC-15, Sigma Aldrich) according to the manufacturer’s instructions. Protein detection was achieved with peroxidase-conjugated secondary antibodies and the ECL system (Amersham Bioscience, Amersham, UK).
9
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About 1 3 10 6 to 2 3 10 7 cells were lysed in ice-cold RIPA buffer (50 mM Tris HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA (pH 8), protease inhibitor cocktail; Sigma). For immunoprecipitations, 1/20 of each sample was saved as an input control and the remaining lysate was incubated with anti-flag M2 agarose beads (Sigma) over night at 4 C on a turning wheel. Beads were washed 4 times with 1 mL of lysis buffer and were mixed with 2x reducing sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT, 0.02% w/v bromophenol blue). Samples were boiled for 6 min and subjected to SDS-PAGE and western blotting onto PVDF membranes. Proteins were detected using primary antibodies against SAFB1, SAFB2, DGCR8 (all Bethyl), DROSHA, beta-actin, GFP, GAPDH (all Cell Signaling Technologies), HA (Roche, Covance) and flag (Sigma) epitope tags. Immunoreactive proteins were visualized with HRP-labeled secondary antibodies (Pierce) and the ECL system (Advansta) on light-sensitive film. Original Western Blot scans have been deposited in Mendeley data (https://dx.doi.org/10.17632/hy8wjhbhz2.1).
10
Western Blot Analysis of RNA Regulatory Proteins
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!