atp5 primary antibody  (Proteintech)

Journal: bioRxiv

Article Title: HSF1 remodels mitochondrial biogenesis and function in cancer cells via TIMM17A

doi: 10.1101/2025.05.12.653547

Figure Lengend Snippet: (A&B) Representative images (A) and quantification (mean ± SD, n=3) (B) of western blot analysis for mitochondrial translation factors, GFM2 and TARS2, in PC3M wild-type cells (PC3M-WT) and PC3M cells with HSF1 knockout (HSF1-KO). The asterisk indicates a higher molecular band recognized by the TARS2 antibody. Unpaired t-test: * P < 0.05; *** P < 0.001. (D) Western blot analysis of TARS2 knockout (TARS2-KO) in PC3M wild-type cells (PC3M-WT) and PC3M cells with HSF1 knockout (HSF1-KO). NTC: non-target control. The asterisk indicates a likely non-specific band recognized by the TARS2 antibody. (D&E) Coessentiality rank and correlation coefficient for all genes with TIMM17A (D) and TIMM17B (E). The positions of mitochondrial translation factors (Mito Translation) in the coessentiality plots are indicated as red dots. (F&G) Representative images (F) and quantification (mean ± SD, n=3) (G) of western blot analysis in PC3M cells following TIMM17A knockout. The asterisk denotes a likely non-specific band recognized by the TARS2 antibody. Either MAPT1 (n=4) or AAVS (n=2) was employed as the negative control in the co-CRISPR assays along with a non-target control (NTC) sgRNA. In each experiment, the tested proteins were normalized to the GAPDH loading control, and the ratios relative to the protein level in NTC were calculated. Results (n=6) were pooled for quantification and plotted into histograms. Unpaired t-test: ns P >= 0.05; ** P < 0.01; *** P < 0.001. (H&I) Representative images (H) and quantification (mean ± SD) (I) of HPG labeling for newly synthesized proteins by mitochondrial translation. HPG labeling was performed in PC3M cells with TIMM17 knockout (TIMM17-KO) or the non-target control (NTC) for 45 min in L-methionine- free medium containing cycloheximide (CHX) to inhibit cytosolic translation. MAPT1 was used as the negative control in the co-CRISPR assay. Immunofluorescence of ATP5 was used as a mitochondrial marker, and DAPI was used to stain DNA. Scare bar: 20 μM. Unpaired t-test: **** P < 0.0001.

Article Snippet: Following one washing step with the Click-iT rinse buffer, MRPS15 or ATP5 primary antibody (Proteintech) was added (1 to 400 dilution) in PBS with 5% BSA and incubated for 1 hour.

Techniques: Western Blot, Knock-Out, Control, Negative Control, CRISPR, Labeling, Synthesized, Immunofluorescence, Marker, Staining

Journal: Advanced Science

Article Title: Phosphorylation of POU3F3 Mediated Nuclear Translocation Promotes Proliferation in Non‐Small Cell Lung Cancer through Accelerating ATP5PF Transcription and ATP Production

doi: 10.1002/advs.202411503

Figure Lengend Snippet: Knockout of POU3F3 downregulated intracellular ATP production by reducing ATP synthase activity. A) Relative ATP synthase activity in WT and POU3F3 knockout H1299 and A549 cells. B) Relative ATP production in WT and POU3F3 knockout H1299 and A549 cells treated with or without ATP synthase inhibitor oligomycin. C) Schematic of the distribution of ATP synthase complex, which are highlighted with different colors on ATP5PF and the subunit associated therewith ATP5 PB and ATP5PD. D) Flag pulldown and western blot to detect the interaction between ATP5PF and ATP5 PB and ATP5PD. E) Western blot detected the interaction between ATP5 PB and ATP5PD in WT and POU3F3 knockout cells.

Article Snippet: Blots were incubated with antibodies against POU3F3 (Proteintech, 18999‐1‐AP, 1:1000 dilution), ERK1/2 (Proteintech, 11257‐1‐AP, 1:1000 dilution), phospho‐ERK1/2 at Thr202/Tyr204 (p‐ERK1/2, Proteintech, 28733‐1‐AP, 1:500 dilution), MEK1/2 (Proteintech, 11049‐1‐AP, 1:1000 dilution), phospho‐MEK1/2 at Ser217/221 (p‐MEK1/2, Cell Signaling Technology, #9154, 1:1000 dilution), ATP5PF (Proteintech, 14114‐1‐AP, 1:1000 dilution), ATP5 PB (Proteintech, 15999‐1‐AP, 1:1000 dilution), ATP5PD (Proteintech, 17589‐1‐AP, 1:1000 dilution), Lamin B1 (Proteintech, 12987‐1‐AP, 1:1000 dilution), HA‐tag (Proteintech, 51064‐2‐AP, 1:3000 dilution), Flag‐tag (Proteintech, 20543‐1‐AP, 1:3000 dilution), Phosphos‐serine (Abcam, ab308512, 1:500 dilution), β‐actin (Proteintech, 66009‐1‐Ig, 1:1000 dilution), GAPDH (Proteintech, 10494‐1‐AP, 1:1000 dilution), Signals were detected, with luminol reagent, using a HRP Substrate Luminol Reagent (Millipore).

Techniques: Knock-Out, Activity Assay, Western Blot

Journal: Advanced Science

Article Title: Phosphorylation of POU3F3 Mediated Nuclear Translocation Promotes Proliferation in Non‐Small Cell Lung Cancer through Accelerating ATP5PF Transcription and ATP Production

doi: 10.1002/advs.202411503

Figure Lengend Snippet: Knockout of POU3F3 downregulated intracellular ATP production by reducing ATP synthase activity. A) Relative ATP synthase activity in WT and POU3F3 knockout H1299 and A549 cells. B) Relative ATP production in WT and POU3F3 knockout H1299 and A549 cells treated with or without ATP synthase inhibitor oligomycin. C) Schematic of the distribution of ATP synthase complex, which are highlighted with different colors on ATP5PF and the subunit associated therewith ATP5 PB and ATP5PD. D) Flag pulldown and western blot to detect the interaction between ATP5PF and ATP5 PB and ATP5PD. E) Western blot detected the interaction between ATP5 PB and ATP5PD in WT and POU3F3 knockout cells.

Article Snippet: For complex V, used ATP5 PB (Abmart, PS18106S, 1:1000 dilution).

Techniques: Knock-Out, Activity Assay, Western Blot