Bca protein assay

Tissues were collected from 10-week-old Dnajb7^+/+ and Dnajb7^−/− males, lysed in RIPA buffer containing protease inhibitor cocktail for 30 min on ice and centrifuged at 13000 rpm for 15 min at 4 °C. The concentration of proteins was determined by the bicinchoninic acid (BCA) protein assay (E11201, Vazyme, China). A total of 20 µg of protein was loaded and separated on 10% SDS–PAGE gels. The primary antibodies used were as follows: anti-DNAJB7 (diluted 1:1000 in TBST, 18540–1-AP, Proteintech, China), anti-DNAJB13 (diluted 1:1000 in TBST, 25118–1-AP, Proteintech, China), anti-Lamin B1 (diluted 1:10000 in TBST, 12987–1-AP, Proteintech, China), anti-GAPDH (diluted 1:10000 in TBST, 60004–1-Ig, Proteintech, China), anti-β-ACTIN (diluted 1:10000 in TBST, P30002M, Abmart, China), and anti-α-TUBULIN (diluted 1:5000 in TBST, 11224–1-AP, Proteintech, China).

For western blot analysis, three tissues from yak at different altitude were
homogenized and lysed using radioimmunoprecipitation assay (RIPA) protein
extraction kit (Solarbio, Beijing, China) and phenylmethanesulfonyl fluoride
(PMSF) (Solarbio, Beijing, China), according to the manufacturer's
instructions. Protein concentrations were quantified using commercial
bicinchoninic acid (BCA) protein assay (Vazyme, Nanjing, China). Protein
samples were separated by 12  $\mathrm{\%}$ sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene
difluoride (PVDF) blotting membrane (Beyotime, Shanghai, China). The
membrane was blocked with 5  $\mathrm{\%}$ non-fat milk in phosphate-buffered saline
(PBS) containing Tween-20 (PBST) for 2  $\mathrm{h}$ at room temperature and then
incubated with rabbit anti-HSP27 polyclonal antibody ( $\mathrm{1}:\mathrm{500}$ ; Bioss, Beijing,
China), rabbit anti-HSP60 polyclonal antibody ( $\mathrm{1}:\mathrm{500}$ ; Bioss, Beijing,
China), and rabbit anti- $\mathit{\beta }$ -actin (loading control) polyclonal antibody
( $\mathrm{1}:\mathrm{5000}$ ; Bioss, Beijing, China) at 4  ${}^{\circ }\mathrm{C}$ overnight. After being
washed with PBST, the membranes were incubated with goat anti-rabbit
IgG/horseradish peroxide (HRP) antibody ( $\mathrm{1}:\mathrm{1000}$ ; Bioss, Beijing, China) for
2  $\mathrm{h}$ at 37  ${}^{\circ }\mathrm{C}$ . After being washed with PBST, the protein signals
were visualized using NcmECL Ultra reagents (New Cell and Molecular Biotech
Co. LTD, Suzhou, China) in an X-ray room.

The CA levels in heart, lung, and longissimus dorsi tissues were detected
using a CA assay kit (Nanjing Jiancheng, Nanjing, China). Specifically, 100  $\mathrm{mg}$ powdered tissues were added to 1000  $\mathrm{\mu }\mathrm{L}$ substrate  $|$ and vortexed
at 0 ${}^{\circ }\mathrm{C}$ for 1  $\min$ , and centrifuged at $\mathrm{11}\mathrm{000}g$ for 10  $\min$ . The
protein concentration of the supernatant was measured using a commercial BCA
protein assay (Vazyme, Nanjing, China). To measure CA levels, 100  $\mathrm{\mu }\mathrm{L}$
supernatant, 700  $\mathrm{\mu }\mathrm{L}$ substrate  $|$ , 100  $\mathrm{\mu }\mathrm{L}$ substrate  $\Vert$ , and 100  $\mathrm{\mu }\mathrm{L}$ substrate  $\vee$ were combined in polyethylene tube (2  $\mathrm{mL}$ ) and
incubated at 37  ${}^{\circ }\mathrm{C}$ for 30  $\min$ . Then, A 200  $\mathrm{\mu }\mathrm{L}$ volume of the
mixture was added to a 96-well plate, and the absorbance values were
measured at 545  $\mathrm{nm}$ on a plate reader (Multiskan FC; Thermo Fisher
Scientific, Beijing, China). The CA concentration was calculated as follows:
$Q=(M-N)/(O-P)\times R/T$ , where $Q$ is the concentration
of CA ( $\mathrm{\mu }\mathrm{mol}{\mathrm{g}}^{-\mathrm{1}}$ tissue), $M$ is the absorbance of the sample, $N$ is the
absorbance of the control, $Q$ is the absorbance of the standard, $P$ is the
absorbance of an empty well, $R$ is the concentration of the standard (0.25  $\mathrm{mol}{\mathrm{L}}^{-\mathrm{1}}$ ), and $T$ is the concentration of the supernatant. Three biological
repeats were measured.