Odyssey irdye

The protocols used for cell lysate preparation and immunoblotting were described previously [42 (link)]. Briefly, 1x10⁸ bacteria were mixed with 100 μl of SDS-PAGE sample buffer (45 mM Tris-Cl, pH 6.8, 10% glycerol, and 1% SDS in the presence or absence of 5% β-mercaptoethanol or 50 mM DTT). A 10 μl sample of cell lysate or 20–40 μl of filtered culture supernatant was separated on a 12.5% polyacrylamide gel under reducing or non-reducing conditions and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore). The blots were blocked with 5% (w/v) non-fat dry milk (NFDM) in PBS containing 0.1% (v/v) Tween 20 (PBST) for 1 h and subsequently incubated overnight at 4°C with an anti-JHP0290 or anti- alkyl hydroperoxide reductase (AhpC) or anti-Urease α (bC-14) (Santa Cruz Biotechnology, SC-22445) antibody in blocking buffer. Protein A affinity-purified polyclonal antibody against JHP0290 and AhpC was generated by EZbiolab (USA). After washing with PBST, the membranes were incubated with a secondary antibody conjugated to the Odyssey IR-dye (Li-COR) for 1 h. The membranes were visualized using an Odyssey IR scanner (Li-COR). The band intensities of the immunoblots were quantified using the ImageJ software (NIH, Bethesda, MA, USA).

Whole hearts were homogenized in RIPA lysis buffer and the extract spun at 13200 g, 10 min, 4 °C. Total cell lysates were electrophoresed and transferred to PVDF membrane and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences). The membrane was incubated with primary antibodies for 2 h at room temperature or overnight at 4˚C, washed in TBST washing solution, and incubated in Odyssey IRDye secondary antibodies (1:5000) for 1 h before visualization with the Odyssey Infrared Imaging System (Li-Cor Biosciences). The primary antibodies used: for detection of LaminA/C (Rabbit, 1:500, Cell Signaling) that is specific to an epitope in the first 50 amino acids in LMNA, mSun1 (mouse mAB, clone 12.11, neat, from B. Burke), GFP (mouse, 1:500, Roche), LaminB1 (rabbit mAB, 1:500, Abcam), anti-HA epitope (rat mAB, 3F10, 1:1000, Roche), GAPDH (rabbit, 1:500, Abcam), and control β-tubulin (mouse, Tub 2.1, 1:1000, Sigma). For detection of the AAV9-DNhSun1 transgene, a mouse mAB specific to the C-terminus of human Sun1 (hSun1, clone 9.1, neat, from B. Burke) was used in combination with protein A conjugated to HRP (1:500, Cell Signaling). GAPDH (rabbit, 1:500, Abcam) was used with anti-rabbit Immunoglobulins/HRP (DAKO, 1:2500) as a loading control. Uncropped and unprocessed scans of blots are in the Source Data file.