Western blot analysis of total cell lysates is performed using antibodies able to recognize either activated or total MAPKs following the manufacturer's instructions (Huang and Tunnacliffe, 2004). T-REx 293 cells are grown and dried as described previously. In order to reduce basal level phosphorylation of ERKs, near confluent cells are incubated overnight in FBS-free medium prior to desiccation or lysis (Z. Huang and A. Tunnacliffe, in preparation).
Protein samples for Western blotting are prepared by direct lysis of cells in 9.6-cm2 wells with equal numbers of starting cells. After complete and careful removal ofmedium by aspiration at the time points ofdesiccation as described earlier, 100 ml of 1 x SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% [w/v] SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% [w/v] bromphenol blue) is added directly to the cell monolayer, and cells are immediately scraped and transferred to a cold microfuge tube on ice. The cell lysates are then sonicated in a sonication bath to reduce viscosity. The sonication is performed at 4° for 10 to 15 s and repeated after 30 s on ice until sample viscosity is reduced. Then the samples are boiled for 5 min and centrifuged at 4° for 15 min at 15,000^. Equal volumes of the super-natants are used for immunoblotting. The lysates can also be stored at -70° until use.
Up to 20 ml of each lysate is loaded onto an SDS-PAGE gel (10 x 10 cm), and the gel-resolved proteins are transferred to nitrocellulose membrane (0.45-mm pore size; Bio-Rad) using a Trans-Blot SD semidry electropho-retic transfer cell (Bio-Rad). Equal loading on SDS-PAGE can be demonstrated by staining gels with Coomassie brilliant blue after transfer and by staining Western blots with Ponceau S solution (which is reversible, i.e., the membrane can be destained) if necessary. The membrane is washed with 25 ml of Tris-buffered saline (TBS; pH 7.6) and blocked with 50 ml of 5% skimmed milk in TBS/T (0.1% Tween-20 in TBS) at room temperature for 1 h. The membrane is washed with TBS/T (25 ml, 3 x 5 min) and incubated overnight at 4° with 10 ml of the primary antibody dilution, which is diluted at 1:1000 (or according to the manufacturer's suggestion) with 5% (w/v) bovine serum albumin (for polyclonal antibodies) or 5% (w/v) nonfat dry milk (for monoclonal antibodies) in TBS/T. After overnight incubation, the membrane is washed again with TBS/T (25 ml,
3 x 5 min) and then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody, which is diluted at 1:1000 (or according to the manufacturer's suggestion) with 1% skimmed milk in TBS/T. After washing the membrane with TBS/T (25 ml, 3 x 5 min), antibody binding is detected using either the ECL Western blotting analysis system (Amersham Biosciences) or the Phototope-HRP Western detection kit (Cell Signaling Technology). After further washing with TBS/T (25 ml,
4 x 5 min), the antibody can be removed from the membrane by incubation in stripping buffer (63 mM Tris, 2% [w/v] SDS, and 0.7% 2-mercaptoethanol) at 50° for 30 min. After washing again with TBS/T (25 ml, 10 x 5 min), the membrane can be reprobed as described earlier for another antibody.
The stripping and reprobing procedure not only saves time and materials, but also greatly facilitates the comparison of the phosphoproteins and their total protein counterparts by eliminating the need to compare separate membrane blots. Because some antigens may be lost with each stripping/ reprobing cycle, it is advisable to probe the antigens expected to be in the least amounts first (e.g., phospho-MAPKs vs total MAPKs). It is also advisable to put membranes into the washing buffer immediately after the final X-ray film exposure. Conditions can be optimized to perform up to five rounds of stripping and reprobing of the same blot.
Relative quantification of gene expression may be analyzed by quantitative real-time PCR as described (Huang and Tunnacliffe, 2004, 2005). Total RNA is prepared using the Cells-to-cDNA II kit (Ambion) following the instruction manual. T-REx 293 cells are grown in 0.5 ml of medium in multidishes (1.9 cm2/well) as described earlier. After complete removal of medium by aspiration from the 1.9-cm2 wells or at the time points of desiccation, 100 ml of ice-cold cell lysis II buffer is added directly to the cell layer. The cells are immediately scraped and pipetted quickly to a cold 1.5-ml microfuge tube in ice. The lysates are processed according to instruction until just before the reverse transcription step, i.e., 75° for 10min, DNase I digestion at 37° for 15min, and 75° for 5 min. RNA preparations can be stored at -20° for a short period or at -80° for a relatively longer time. Reverse transcription is also performed using the kit according to instructions with the random decamers provided and 5 ml of cell lysate (RNA). Resultant cDNA can be diluted 10 to 20 times with water and stored at -20° for a short time.
Relative quantification ofcDNA by real-time PCR is performed using a Rotor-Gene real-time cycler (Corbett Research) and QuantiTect SYBR Green PCR kit (Qiagen). The critical threshold values are used to calculate the relative amounts of cDNA according to the delta-delta method (Pfaffl, 2001). ACTB (b-actin) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) are used as reference gene transcripts. Whenever appropriate the primers for target genes are designed to span exon-exon junctions to avoid amplification from potential contaminating genomic DNA (although RNA preparations are digested with DNase I). Primers are also chosen so that the size of the resulting amplicons is 50 to 150 bp. Specific amplification of transcripts is verified by gel electrophoresis, where only one DNA fragment should be observed, and by melt curve analysis of the real-time PCR products, where a single peak should be seen. Typical primer sequences are given in Huang and Tunnacliffe (2004, 2005).
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