Quantity one 1 d software

Western blot images were quantified using Quantity One 1-D software Version 4.6.8 (BIO-RAD; Hercules, CA, USA). All data are presented as the mean ± SEM. The statistical analysis was conducted using an Analysis of Variance (ANOVA), with the Tukey–Kramer post hoc test employed to assess the significance of differences between the experimental groups. A p-value less than 0.05 was deemed to indicate statistical significance.

Samples were processed according to the protocol described elsewhere [47] (link). Briefly, following supplementation of samples with BN-sample buffer, the molecular weight marker (NativeMARK Unstained Protein Standard, Life Technologies) and 30 µg of samples were loaded into polyacrylamide gels and run at 80V at 4 °C. The gel containing the proteins of interest was eletrotransferred to a PVDF membrane (Hybond P 0.5 µm, Amersham) for 2 h at 0.2A, at 4 °C. Afterwards, membranes were incubated with monoclonal primary antibodies [anti-NDUFA9 for CI; anti-SDHA for Complex II (CII); anti-UQCRC2 for Complex III (CIII); anti-COX IV for CIV; and anti-ATP5A for Complex V (CV)]. Membranes were incubated with the anti-mouse HRP-conjugated secondary antibody solution. Subsequently, detection was carried out using a chemiluminescence substrate (Clarity Western ECL Substrate, Bio-Rad), through the ChemiDocTM XRS+ System (Bio-Rad). Protein band intensities were calculated by Quantity One® 1-D software (Bio-Rad) from at least 3 independent experiments. Relative semi-quantitation of each complex assembled was performed in comparison to CII levels.

The collected 1-ml sample of the fermentation broth was centrifuged for 10 min at 12,000 rpm, and the supernatant was removed. The cells were resuspended in 1 ml of 20 mM Tris-HCl buffer (pH 7.5) and lysed using an Ultrasonic Cell Disruptor (operating for 5 s, pausing for 5 s, 40 times). The cell lysate was centrifuged at 4°C, 12,000 rpm for 10 min to remove the cell debris. Eighty microliters of the supernatant was mixed with 20 μl of 5 × SDS-PAGE loading buffer and heated to 100°C in a metal block for 10 min. The final protein samples were used for SDS-PAGE (WSHT, China).
To facilitate the gel analysis, we calculated normalized loading volumes for standard gels (Novagen). Samples containing the protein from an amount of cells corresponding to 0.15 OD₆₀₀ units (10-well gel) or 0.075 OD₆₀₀ units (15-well gel) were added to the corresponding protein gel wells and separated at 90 V until the dye reached at the end of the gel. Quantitative comparison was conducted using Quantity One 1-D software (BioRad, United States) and Quantity One User Guide for Version 4.6.2. According to the calculation results of Quantity One, N_Vn represents the value of the GOI overexpression band in VnDX, and N_Ec represents the value of the GOI overexpression band in BL21(DE3). VnDX = BL21(DE3) when the following inequality is satisfied. GraphPad prism software was used for statistical analysis of data.

Proteins from the cellular fraction of NPAs were extracted from QIAzol using chloroform-isopropanol and 1% SDS. Proteins were quantified by BCA (Thermo Fisher Scientific) and resolved (10 µg) in Acrylamide/Bis-acrylamide gels. They were then transferred to polyvinylidene difluoride (PVDF) Amersham HybondTM-P membranes (GE Healthcare, Buckinghamshire, UK) and blocked 2 h in 1× PBS/5% non-fat-dried milk/0.2% Tween-20 at room temperature. Overnight incubation was performed at 4 °C with antibodies against β-actin (Cell Signaling Technology Cat# 4970, RRID:AB_2223172, Leiden, The Netherlands, 1:1000) and filaggrin (Novus Biologicals, LLC, CO, USA, 1:500) in 1× PBS/0.5% non-fat-dried milk/0.2% Tween-20, and further incubation with the secondary anti-rabbit (Millipore Cat# AP156P, RRID:AB_11213985, Burlington, MA, USA, 1:1000) or anti-mouse (Thermo Fisher Scientific, 1:1000) antibody-HRP for 2 h at room temperature. Immobilon^® Crescendo Western HRP Substrate was used, with visualization using an Amersham Imager 600 (GE Healthcare). Data were curated using Quantity One 1-D software (Bio-Rad).