Fig. 3

Purified AK2 specifically bound to rhCDK4. a CDK4-binding activity of purified AK2. Microtiter plates were coated with rhCDK4 or irrelevant proteins (rhPHD2, rhCOX-2 or BSA) then wells were incubated sequentially with purified AK2, anti-V5 tag antibody then HRP-conjugated IgG to conduct the ELISA assay. Wells lacking purified AK2 served as the negative control (NC) (*** P < 0.001 vs. corresponding irrelevant protein controls or NC group, # P > 0.05 vs. corresponding irrelevant protein controls). b AK2 competition with anti-CDK4 polyclonal antibody for CDK4 binding. Wells coated with rhCDK4 were incubated with AK2 that had been pre-incubated with irrelevant scFv AD5, anti-CDK4 mouse monoclonal antibody (PC group) or BSA (NC group) in order to determine whether AK2 could competitively bind to CDK4 in the presence of anti-CDK4 rabbit polyclonal antibody. The ELISA assay was then conducted by sequential probing with anti-CDK4 rabbit polyclonal antibody then HRP-conjugated goat anti-rabbit IgG followed by the addition of HRP chromogenic solution (*** P < 0.001 vs. corresponding pre-incubation with irrelevant scFv AD5 or NC group, # P > 0.05 vs. NC group). c Specificity of binding of AK2 to CDK4. Purified AK2 that had been pre-incubated with different concentrations of CDK4 or with other irrelevant proteins was added to wells coated with rhCDK4 in order to assess specificity of binding of AK2 to CDK4. The absorbance at 492 nm was measured after sequential addition of anti-V5 Tag monoclonal antibody, HRP-conjugated goat anti-mouse IgG then HRP chromogenic solution (*** P < 0.001 vs. corresponding pre-incubation with different concentrations of CDK4, # P > 0.05 vs. corresponding pre-incubation with 3.00 μg and 4.00 μg of CDK4, & P > 0.05 vs. corresponding pre-incubation with other irrelevant proteins). Abbreviations: rhCDK, recombinant human cyclin-dependent kinase; rhPHD, recombinant human prolyl hydroxylase; rhCOX, recombinant human cyclooxygenase; BSA, bovine serum albumin; IPTG, isopropyl β-D-1- thiogalactopyranoside