To evaluate the phosphorylation state of DARPP-32 in our experimental conditions, striatal slices were incubated with Roscovitine 20 μM or vehicle for 10 min in artificial brain fluid. After incubation, striatal tissue was homogenized in lysis buffer containing 26 mM Tris–HCl, 1% (v/v) Triton X-100, 1.3 M glycerol and 130 mM NaCl, with the protein phosphatase inhibitor cocktail Complete mini tab (Roche). The samples were recollected and centrifuged for 5 min at 4500 rpm; supernatants were recollected and store at −70°C. Protein quantification was made using the Bradford method and 80 μg of protein was loaded in 10% (w/v) PAGE for electrophoresis. Proteins were transferred to PVDF membranes and incubated with primary antibodies against pDARPP-32 Trh34 (1:1000), pDARPP-32 Trh75 (1:1000) and Actin (1:1000) for 12–20 h at 4°C, and with secondary antibodies (1:2000) with HRP for 2 h at room temperature. Protein detection was performed through the use of luminol with a chemiluminescence method (Millipore); images were acquired via a scan (HP Scanjet G2410) and analyzed with Image J software for the evaluation of densitometry.
Complete minitab
Manufactured by Roche
Sourced in United Kingdom, Canada
Complete Minitab is a statistical analysis software package designed for data analysis, process improvement, and quality management. It provides a comprehensive set of tools for data exploration, hypothesis testing, regression analysis, and quality control charting.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence method, by Merck Group (1 mentions)
Anti rac1, by BD (1 mentions)
Anti β actin, by Abcam (1 mentions)
Complete phostop, by Roche (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab8226, by Abcam (1 mentions)
Alx 215 049 r100, by Enzo Life Sciences (1 mentions)
Alexa fluor 647 goat anti rabbit igg a21245, by Thermo Fisher Scientific (1 mentions)
Chloroquine diphosphate, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
5 protocols using complete minitab
1
Quantifying DARPP-32 Phosphorylation in Striatal Slices
2
Artery Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Arteries were homogenized in Tris‐TX‐100 buffer (25 mmol/L Tris pH 7.4, 5 mmol/L EDTA, 0.2% Triton X‐100, protease inhibitors [Complete mini‐tab; Roche, Welwyn Garden City, UK], 1 mmol/L sodium orthovanadate, 200 μmol/L sodium pyrophosphate) on ice. The homogenate was incubated at 4°C for 30 min, centrifuged at 800g for 10 min at 4°C to pellet nuclei and cell debris and the protein concentration of the supernatant determined by Bradford assay. The sample volume was adjusted with Tris‐TX‐100 buffer to a final concentration of 1 mg/mL. An aliquot was removed for sphingomyelinase assay and to the remainder Laemmli sample buffer was added and the sample stored at −20°C.
3
Extraction and Quantification of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate cell lysates, culture medium was aspirated, and cells were washed with ice‐cold PBS. Cell lysis buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 1mM EDTA, 1mM EGTA, 1% Triton X‐100, protease inhibitor cocktail: Complete Minitab, and phosphatase inhibitor cocktail: Complete PhoStop (Roche, Laval, Canada)) was then added to cells, and they were frozen at −20°C. Total protein was quantified using the Precision Red Protein Assay (Cytoskeleton, Denver, CO), and protein concentration was normalized between samples. Western blots were performed on normalized protein extracts from Caco‐2 intestinal epithelial cells. Membranes were probed with the appropriate primary antibody (anti‐RAC1, BD Bioscience #610651; anti‐β‐actin, Abcam #mAbcam‐8226) and corresponding horseradish peroxidase (HRP)‐conjugated secondary antibody. Membranes were imaged using the MicroChemi Bio‐Imaging system.
4
Western Blot Analysis of hERG Expression
5
IGF-1 and EGF Signaling Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were serum-starved for either 2 hrs or overnight in media containing 0.1% BSA and then stimulated with IGF-1 (100 ng/ml) or hEGF (50ng/ml) for the time periods indicated in the Figures. To inhibit internalization, cells were incubated with 10ug/ml chlorpromazine for 1 hour prior to and during stimulation. Cells were infected with lentiviruses containing shBECN1-resistant Flag-BECN1 for over-expression and rescue experiments and stimulated 48hrs post-infection. Cells were extracted at 4°C for 10 minutes in a 20 mM Tris buffer, pH 7.4, containing 0.15 M NaCl, 1% NP-40, 10% Glycerol, 1 mM sodium orthovanadate, 10 mM NaF and protease inhibitors (Complete Mini Tab; Roche). Cell extracts were immunoblotted as described previously48 (link). Bands were detected by chemiluminescence using a ChemiDoc XRS+ system (BioRad Laboratories) and band intensities were quantified by densitometry using Image Lab (Beta 1) (Bio-Rad Laboratories). PI3P lipid levels were measured using the PI3P Mass ELISA Kit (Catalog # K-3300) according to manufacturer's instructions.
For autophagic flux assay, MDA-MB-231 cells expressing shGFP or shATG5 were incubated in either RPMI 1640 supplemented with 10% fetal bovine serum or Hank's Balanced Salt Solution (GIBCO®), with and without 25 μM Chloroquine diphosphate (Catalog # C6628; SIGMA) for 4 hours.
For autophagic flux assay, MDA-MB-231 cells expressing shGFP or shATG5 were incubated in either RPMI 1640 supplemented with 10% fetal bovine serum or Hank's Balanced Salt Solution (GIBCO®), with and without 25 μM Chloroquine diphosphate (Catalog # C6628; SIGMA) for 4 hours.
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!