Bradford protein concentration assay

Total protein was isolated from homogenized extrinsic tongue muscles of adult mice using methods previously described (Adreani et al., 2006 (link)). Following sample protein quantification through a Bradford protein concentration assay (Thermo Fisher Scientific), MyHC profiles of extrinsic tongue muscles were analyzed through separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Adreani et al., 2006 (link)), and silver staining to visualize protein isoform bands. Stained gels were digitally scanned using a flatbed scanner and band signal intensity was quantified using UN-SCAN-IT gel analysis software (Silk Scientific). Each mouse muscle sample was analyzed in duplicate.

Following the protocol described by Bichet and colleagues [32 (link)], confluent MDCK-I or A549 cell lines, for KAM-1325 or KAM-2000 microarrays, respectively, were incubated with T4 phages or Filter control samples for 8 hours at 37°C and 5% CO₂. After incubation, the cells were scraped in lysis buffer before sonication. All samples were treated as chemically lysed proteins and followed the recommended protocol by Kinexus. The MDCK-I and A549 proteins were quantified using the Bradford protein concentration assay (Thermo Fisher Scientific). The MDCK-I samples were then incubated on the KAM-1325 array before sending the array to Kinexus for analysis, while the A549 samples were sent directly to Kinexus for analysis on the KAM-2000 array. Briefly, microarray datasets were filtered to remove low signal intensity and/or relatively high error signals compared to control signals. The network analysis was then separately run for both up- and down-regulated phosphorylation events before being assembled into a comparative pathway map [45 (link)]. Only pathways with more than 2 intermediates and with fold-changes greater than 5% CFC (% changes from control) were selected for further consideration.