P-Actin

Two subsequent studies by Kauret al. (1992) and McLain et al. (1992) used the same p-actin promoter to drive misexpression of the Hox-2.2... 

Zhang HL, Byrd AL, Singer RH, Bassell GJ. 1999. Neurotro-phin regulation of P-actin mRNA and protein localization within growth cones. J. Cell Biol. 147 59-70. 

P-Actin promoter amplification P-Actin nested primers for second-round amplification in QM-MSP=... 

Fig. 9.4 Establishment of the quantitative methylation-specific polymerase chain reaction (MSP) analytical procedure. The ABI PRISM 7900 HT Sequence Detector system was used to perform real-time polymerase chain reaction (PCR) using MSP primers and bisulfite-modified template DNA. Upper panels Setting up the conditions to obtain the standard curves with 50% (A) or 25% (B) sequential dilution of the template. Lower panels The amplification curves on the left represent P-actin, unmethylated, and methylated MSP products, respectively, for reelin (RELN) (C). Amplification curves were compared at the set threshold before 40 cycles. Amplification curves from various samples are shown in the lower panel right (D)...

Normalize the C.j, of unmethylated or methylated product with the C.,. of the P-actin gene, amplified in separate reactions for quantitation (see also Note 4). 

Figure 6. Comparative gene expression ratios in ARF kidneys of MSC- and vehicle-treated animals. Data were generated by referencing each gene to p-actin as internal control. A MSC treatment cansed significant (P < 0.05) suppression (> 10 fold) of proinflammatory IL-ip, TNF a, and IFN-y (above bars actnal valnes). Anti-inflammatory lL-10 was robustly expressed in MSC-and not in vehicle treated animals. Filled bars on all panels depict gene expression ratio of 1, i.e., a value obtained when gene expression ratios between MSC- and vehicle-treated animals are "equal. B MSC treatment cansed increased gene expression of bFGF and TGF-a, whereas that of HGF was suppressed. C antiapoptotic Bcl-2 expression was robnstly indnced, whereas that of inducible nitric oxide synthase (iNOS) was snppressed in MSC- vs. vehicle-treated animals. eNOS, endothelial NOS.

Primary antibody Monoclonal anti-GAPDH antibody and monoclonal anti-p-actin antibody from mouse (see Note 1). 

Probing with Another Primary Antibody (Anti-p-Actin)... 

To detect another protein expression, the membrane is rinsed twice with TEST and incubated with another primary antibody (anti-p-actin) for 1 h at room temperature on shaking. 

Since the molecular weight of GAPDH is around 36 kDa and P-actin is about 42 kDa, their bands appear in distinct areas of the membrane and are unlikely to interfere with each other. However if the two or more proteins to be detected on the same membrane have a similar molecular weight, stripping and re-probing are necessary (see Note 15). 

Fig. 4. Western blot image of GAPDFI expression in A549 cells at 72 h post transfection with LAFI4-L1/siRNA complexes. The intensity of GAPDFI protein band decreases as the amount of siRNA per well increases. For the negative control (-ve), 100 pmol siRNA/well was used. p-Actin was used as internal control.

To calculate the gene silencing efficiency of the peptide/siRNA complexes, the expression of the protein of interest (in this case GAPDH) has to be first normalized with the expression of P-actin (protein for internal control) of the same sample. 

FIGURE 3.9 Western blotting analysis of uncoupling protein 1 (UCP1) in epididymal WAT and relative expression level of UCP1 protein compared to p-actin (Okada etal., 2011). Columns with different superscript letters are significantly different (p<0.05). 

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