Journal:

Article Title: Polo-like kinase 1 (Plk1) in non-melanoma skin cancers

doi:

Figure Lengend Snippet: A) Plk1 protein expression. NHEK and human squamous cell carcinoma A253 and A431 cells were trypsinized, washed with ice-cold PBS and lysed for protein with RIPA buffer plus PMSF and protease inhibitor cocktail (Pierce, IL) for immunoblot analysis. Briefly, 30 μg of protein was subjected to SDS-PAGE and transferred onto nitrocellulose membrane. Blots were blocked followed by probing with Plk1 primary antibody (Upstate, MA) and then appropriate HRP-conjugated secondary antibodies followed by enhanced chemiluminescent detection. Blots were subsequently stripped and re-probed with goat anti-β-actin (Santa Cruz, CA) primary antibody followed by appropriate secondary and chemiluminescent detection as a loading control. The quantitation of protein was performed by digital analyses of protein bands (TIFF images) using UN-SCAN-IT software (Silk Scientific, Inc., UT) and the data represented as relative Plk1 protein levels +/- standard deviation of three experiments. All comparisons to NHEK expression were statistically significant with P-values < 0.01 using two-tailed Student’s T-test; B) Plk1 mRNA expression. RNA was isolated using Trizol reagent (Invitrogen, CA), treated with DNAse (Promega, WI), and first strand cDNA created with M-MLV reverse transcriptase (Promega, WI) according to vendor’s protocol. Quantitative real time RT-PCR was performed in triplicate in 20 μl reactions with SYBR® Premix Ex Taq™ Perfect Real Time (Takara, WI) with 50 ng first strand cDNA and 0.2 μg each Plk1 forward (5’-CCCCTCACAGTCCTCAATAA-3’) and reverse (5’-AATAGTCCACCCACTTGCTG-3’) primers or 0.2 μg each GAPDH forward (5’-GGGTGTGAACCATGAGAAGT-3’) and reverse (5’-GTAGAGGCAGGGATGATGTT-3’) primers. Data is expressed as relative Plk1 mRNA using the ΔΔCT method with GAPDH as an endogenous control +/- standard deviation for three separate experiments. *P-value < 0.01; C) Protein expression of Cdk1, Cdc25C or Cyclin B1. NHEKs, A253 and A431 cells were collected, lysed and analyzed using immunoblot analysis as described above. Blots were probed for Plk1 targets viz. Cdk1, Cdc25C or Cyclin B1 using appropriate antibodies (Cell Signaling, MA) and β-actin as loading control. Relative protein expression was quantitated as previously and presented as relative target protein levels +/- standard deviation of three separate experiments. All comparisons to NHEK expression were statistically significant with P-values < 0.01 using two-tailed Student’s T-Test.

Article Snippet: NHEK and human squamous cell carcinoma A253 and A431 cells were trypsinized, washed with ice-cold PBS and lysed for protein with RIPA buffer plus PMSF and protease inhibitor cocktail (Pierce, IL) for immunoblot analysis.

Techniques: Expressing, Protease Inhibitor, Western Blot, SDS Page, Membrane, Control, Quantitation Assay, Software, Standard Deviation, Two Tailed Test, Isolation, Reverse Transcription, Quantitative RT-PCR