The largest database of trusted experimental protocols

2 protocols using egr1 c19

1

Western Blot Analysis of Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
MDA-MB 231 and SUM159 cells were grown in 10 cm dishes, exposed to the treatments and then lysed as previously described [48 (link)]. Equal amounts of whole protein extract were electrophoresed through a reducing SDS/8 and 10% (w/n) polyacrylamide gels, electroblotted onto a nitrocellulose membrane (Amersham Biosciences, GE Healthcare, Milan, Italy), and probed with primary antibodies against Y397-FAK (Cell Signaling Technology, Milan, Italy), FAK (H-1) (Santa Cruz Biotechnology, DBA, Milan, Italy), phosphorylated ERK1/2 (E-4) (Santa Cruz Biotechnology, DBA, Milan, Italy), ERK2 (C-14) (Santa Cruz Biotechnology, DBA, Milan, Italy), p-AKT1/2/3 (Ser 473)-R (Santa Cruz Biotechnology, DBA, Milan, Italy), AKT/1/2/3 (H-136) (Santa Cruz Biotechnology, DBA, Milan, Italy), c-FOS (H-125) (Santa Cruz Biotechnology, DBA, Milan, Italy), EGR1 (C19) (Santa Cruz Biotechnology, DBA, Milan, Italy), CTGF (Origene, DBA, Milan, Italy) and β-actin (C2) (Santa Cruz Biotechnology, DBA, Milan, Italy). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, DBA, Milan, Italy) and then revealed using the ECL™ Western Blotting Analysis System (GE Healthcare, Milan, Italy).
+ Open protocol
+ Expand
2

Western Blot Analysis of Protein Targets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Western blot analysis of protein targets was performed as previously described (Tai et al. 2002 (link)(Tai et al. , 2007 (link)(Tai et al. , 2009)) (link). Briefly, 50 mg of total protein per sample was resolved on 12% SDSpolyacrylamide gels and subsequently transferred to PVDF membranes (BioRad) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol) at 100V for 1 h. Transfer and equal loading of protein were verified by Ponceau S stain (Sigma). Membranes were incubated for 1h at 48C in blocking solution containing 5% skim milk in Tris-buffered saline with Tween-20 (TBST; 10 mM Tris-HCl, 150mM NaCl, 0.05% Tween-20), then rinsed in TBST (3!10 min) before overnight incubation with primary antibodies specific for rat PNMT (Immunostar, Hudson, WI, USA; 1:3000 dilution), Egr-1 (C-19, Santa Cruz Biotechnologies; 1:1000), Sp1 (PEP-2, Santa Cruz; 1:2500), GR (M-20, Santa Cruz; 1:2000), AP-2 (Millipore, Billerica, MA, USA, 1:2000) in 5% skim milk-TBST. Membranes were then rinsed in TBST (3!10 min) and incubated with HRP-conjugated secondary IgG (Sigma; 1:5000) in 5% skim milk-TBST for 1 h, followed by a final series of rinses in TBST (3!10 min). Proteins were then detected by enhanced chemiluminescence (Haan & Behrmann 2007) (link) and subsequent exposure to film (Pierce, Rockford, IL, USA).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!