Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones. Kityk et al.

Figure S2: Immunoblot analysis of the expression of wild type and mutant dnaK. The ∆dnaK52 strain (BB1553) carrying a lacIq plasmid was transformed with plasmids encoding mutant and wild-type dnaK under the control of an IPTG-inducible promoter. Cells were grown under the conditions used in complementation assays (50 µM IPTG, 40 °C), extract prepared, separated by SDS-PAGE and DnaK detected by immunoblotting using a DnaK-specific polyclonal antiserum. Lanes from left to right: pre-stained molecular weight marker, extract of cells expressing dnaKwt, vector control, dnaK-Q378A, dnaK-T420A, dnaK-D477A, empty lane.图S2：野生型与突变型dnaK的表达免疫印迹分析。携带lacIq质粒的ΔdnaK52菌株（BB1553）经转化导入了由异丙基-β-D-硫代半乳糖苷（IPTG）诱导型启动子调控的、分别编码突变型与野生型dnaK（dnaKwt）的质粒。菌体在互补实验设定的培养条件（50 μM IPTG，40 ℃）下培养，随后制备菌体提取物，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，使用DnaK特异性多克隆抗血清通过免疫印迹法（immunoblotting）检测DnaK蛋白。泳道自左至右依次为：预染分子量标准品、表达野生型dnaK（dnaKwt）的菌体提取物、空载体对照、dnaK-Q378A、dnaK-T420A、dnaK-D477A，以及空白泳道。