*4.7. Western Blotting and Immunodetection*

Whole-cell extracts were loaded onto 10% polyacrylamide gels, electrophoresed, and blotted on polyvinylidene difluoride membranes (Bio-Rad Laboratories, Munich, Germany). The membranes were blocked with 5% nonfat dry milk in phosphate buffered saline (PBS) containing 0.1% Tween-20 and incubated overnight with the corresponding primary antibody. The primary and secondary antibodies used in this study are shown in Table 2. Membranes were incubated with corresponding secondary antibodies for 1 h. ECL-Plus detection was performed according to the manufacturer's instructions (GE Healthcare, Little Chalfont, UK).


#### *4.8. RT-qPCR*

The relative expression levels of selected genes were studied using RT-qPCR. Total RNA was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich), and RNA concentration and purity were determined spectrophotometrically. For all samples, equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic). RT-qPCR was carried out in 10 μL reaction volumes using the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and analyzed using the 7500 Fast Real-Time PCR System and 7500 Software v. 2.0.6 (both Life Technologies, Carlsbad, CA, USA). Changes in the transcript levels were determined using the 2−ΔΔ*C*<sup>T</sup> method [33]. The housekeeping gene *HSP90AB1* was used as an endogenous reference control. The primers used in this study are listed in Table 3.


F, forward primer; R, reverse primer.
