Journal: Cancer Biology & Therapy

Article Title: EGFR signaling defines Mcl - 1 survival dependency in neuroblastoma

doi: 10.1080/15384047.2014.1002333

Figure Lengend Snippet: Mcl-1 dependent NB cells NLF (A) and SK-N-BE(2) (B) were exposed to EGFR inhibitors, erlotinib or cetuximab, and ABT-737 simultaneously and evaluated after 48 hours for changes in survival by WST-1. (C) NLF cells were exposed to given concentrations of U0126, erlotinib, or LY294002 for 24 hours, harvested for protein, and evaluated for changes in EGFR signaling and Bcl-2 family protein expression. (D) Protein from an Mcl-1 co-IP was treated with Lambda Protein Phosphatase (LPP) and then evaluated for changes in Bim:Mcl-1 interactions by immunoblot.

Article Snippet: 4 The Mcl - 1 protein;Mcl - 1 antibody matrix was then pelleted with centrifugation, and resuspended in a lambda phosphatase (LPP, Promega, Sweden) containing solution (4 uL LPP:4 uL 10X Buffer: 32 uL co-IP solution + 1 M DTT (Millipore)) and incubated at 37°C for 40 min. An equal portion of Mcl - 1 protein;Mcl - 1 antibody matrix was resuspended in IP buffer alone (without LPP) and incubated at 37°C for 40 min to control for the effects of heating on protein interactions.

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Protein kinase C controls activation of the DNA integrity checkpoint

doi: 10.1093/nar/gku373

Figure Lengend Snippet: Analysis of Pkc1 electrophoretic mobility after induction of DNA damage. ( A ) Left panel: exponentially growing wild-type cells expressing a GFP-tagged Pkc1 protein (JCY1511) were incubated for 2 h in the absence or presence of 0.2 M HU; right panel: raffinose-grown wild-type (JCY1511) and GAL1:HO (JCY1514) cells expressing a GFP-tagged Pkc1 protein were incubated for 4 h in the presence of 2% glucose or 2% galactose. Electrophoretic mobility of Pkc1-GFP was investigated by western analysis using an anti-GFP antibody. An unspecific band in the western blot or the ponceau staining of the membrane is showed as a control of migration. Activation of the checkpoint kinase Rad53 is showed as a control of DNA damage induction. ( B ) Raffinose-grown GAL1:HO (JKM139) cells were incubated for 4 h in the presence of 2% glucose or 2% galactose. Electrophoretic migration of endogenous Pkc1 was investigated by western analysis using an anti-yeast Pkc1 antibody. ( C ) Raffinose-grown GAL1:HO (JKM139) and GAL1:HO tel1 (JCY1654) cells were incubated for 4 h in the presence of 2% glucose or 2% galactose. Electrophoretic migration of endogenous Pkc1 after incubation of cell extracts in the absence or presence of λ protein phosphatase was analyzed as in (B). ( D ) Electrophoretic migration of Pkc1-GFP in wild-type (W303-1a) mec1 (JCY1580) and tel1 (JCY1583) cells after addition of HU was analyzed as described in (A). ( E ) Raffinose-grown GAL1:HO (JKM139) and GAL1:HO tel1 (JCY1654) cells transformed with pPKC1-GFP plasmid were incubated for 4 h in the presence of 2% glucose or 2% galactose. Electrophoretic migration of Pkc1-GFP was investigated by western analysis using an anti-GFP antibody.

Article Snippet: The dephosphorylation assays were carried out by incubating 10 mg of cell extract in λ protein phosphatase buffer (50 mM Tris-HCl pH 7.8, 5 mM DTT, 2 mM MnCl2, 100 μg/ml bovine serum albumin) containing 200 U of λ protein phosphatase (MERCK Millipore) for 20 min at 30°C.

Techniques: Expressing, Incubation, Western Blot, Staining, Membrane, Control, Migration, Activation Assay, Transformation Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: Detection of the Cell Cycle-Regulated Negative Feedback Phosphorylation of Mitogen-Activated Protein Kinases in Breast Carcinoma using Nanofluidic Proteomics

doi: 10.1038/s41598-018-28335-8

Figure Lengend Snippet: Identification of protein isoforms of the MAPK pathway using capillary isoelectric focusing (cIEF) immunoassays. ( a ) Western blotting with antibodies directed against MEK1, MEK2, and ERK1/2 proteins using total cell extracts (TCEs) of the breast cancer cell line BT474. TCEs of the BT474 cell line were either untreated (−λ) or treated (+λ) with λ phosphatase prior to Western blot assays. β-actin served as a loading control. Cropped Western blots were from different gels loaded with the same amount of TCEs. All gels were run on the same day and subjected to the same experimental procedures, including the same exposure duration during detection. A representative immunoblot of β-actin is presented to highlight comparable TCE loading between lanes. M.W.: molecular weight. ( b–d ) Raw cIEF immunoassays of ( b ) MEK1, ( c ) MEK2, and ( d ) ERK1/2 using TCEs of BT474 cell line without (−λ) or with (+λ) λ phosphatase treatment. Specific antibodies directed against both ERK1 and ERK2 isoforms (ERK1/2), the ERK1/2 phosphor-isoform (p-ERK1/2), the ERK1 isoform, or the ERK2 isoform were used to resolve ERK1/2 isoforms. Graphical presentations of cIEF immunoassays in b–d for ( e ) MEK1, ( f ) MEK2, ( g ) ERK1/2, ( h ) pERK1/2, ( i ) ERK1, and ( j ) ERK2. The peak intensities were normalized to 1 for all cIEF immunoassay data. pI: isoelectric point.

Article Snippet: Approximately 1 μl of λ phosphatase (cat. no. 14–405; Merck Millipore, Billerica, MA) was added to 1 μl of reaction buffer (final concentrations of 5 mM DDT, 50 mM Hepes, 100 μM EDTA, 2 mM MnCl 2 ) and 8 μl of TCEs (2 mg/ml).

Techniques: Western Blot, Control, Molecular Weight