Trans-activation of the p21 promoter is necessary but not sufficient to increase p21 expression. A, HeLa/18 × 21-EGFP cells were transfected with p21Δ1-Luc without or with 3 μg of each of the expression plasmids for wild-type and mutant Tax (M22, M47, L235F, A108T, V89A) as in Fig. 1. The reporter assays and analyses are as in Fig. (1). B, Twenty micrograms of total cell proteins from each transfection were resolved in a 12%SDS-polyacrylamide gel and immunoblotted using the 4C5 mouse hybridoma antibody against Tax, the rabbit anti-p21 antibody, and the rabbit anti-β-actin antibody respectively. C, A Summary the biological properties of various Tax alleles. NF-κB, LTR, and p21p-Luc fold transactivation shows the fold activation of the E-selectin-Luc, HTLV-1 LTR-Luc, and the p21 proximal promoter-Luc reporters by wild-type tax (Tax) or the respective mutant allele (M22, M47, L235F, A108T, and V89A) is indicated. APC % G1: The fractions of HeLa cells in G1 after transduction by wild-type tax (Tax) or mutant tax allele (L235F and A108T). ND denotes "not determined". p21 protein arbitrary unit indicates the ratio of the level of p21 protein normalized again that of Tax after densitometer scanning. The p21 level in M47 is likely underestimated because of the non-linearity of film over-exposure to the immunoblot. The mRNA of p21 is significantly stabilized by M47 as shown in Fig. 6.