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Using SDS-PAGE analysis of culture medium from SEAP-AcMNPV infected Ao38 cells, we identified an intensely staining band of approximately 64 kDa (Fig. 6).
The relative intensities of positive staining band for collagen II were 54.2 ± 4.4% and 76.2 ± 8.6% after 1 week and 2 weeks, respectively.
The relative intensities of positive staining band for TGF- β3 were 46.5 ± 7.4% and 75.9 ± 6.8% after 1 week and 2 weeks, respectively.
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Using automated software (LI-COR Biosciences) densitometry was performed to obtain the integrative intensity of all positively staining bands.
After ponceau staining, bands corresponding to LRRK2 or 4E-BP were excised according to their molecular weight and incorporated radioactivity was assessed by LS 6500 mulitipurpose scinitillation counter (Beckman coulter, Sykesville, MD).
Gelatinolytic activity was visualized as negatively staining bands.
For example, regions of the gel with intensely staining bands will have high local contrast, whereas other regions with few bands will have low contrast.
However, 8 archived samples showed faintly staining bands for mitochondrial 16S rRNA gene amplicons, suggesting loss of DNA integrity in these older samples.
The recombinant purified PepP contained no activity stained band at 200 kDa.
However, performing normal SDS-PAGE analysis revealed within all samples, one Coomassie stained band with an apparent molecular weight in the range of 45 kDa (data not shown).
Mass Spectrophotometric analysis of the protein present in coomassie stained band in both wild type and PknJ-KD-K43A also validated it to be PknJ.
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