Ponceau red

S. aureus cell pellets were homogenized with lysing matrix B (MP Biomedicals; 100 mg beads/ml cells) in 25 mM Tris (pH 7.5) on a Retsch MM400 mixer mill at 15 Hz in four 3-min cycles. Clarified lysates were recovered by spinning at 20,817 × g at room temperature for 5 min to remove cell debris. A total of 0.1 to 0.2 A₂₈₀ units of cell lysate were analyzed on 4% to 20% TGX SDS-PAGE gels (Bio-Rad), and the proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo system (Bio-Rad). The membrane was stained with Ponceau red (Amresco) to ensure equal loading, followed by immunoblotting using anti-Rnr (1/1,000 dilution), anti-S11 (1/4,000 dilution), anti-HPF (1/4,000 to 1/8,000 dilutions), and anti-FLAG (1/1,000) antibodies. Polyclonal rabbit anti-S11 (25 (link)) and anti-HPF (43 (link)) antibodies were generated and described previously. Anti-FLAG M2 was from Cell Signaling (catalog number 2368). To generate anti-Rnr antibody, two peptides corresponding to residues (234 to 257 and 576 to 595) of the S. aureus RNase R (Cys-²³⁴QEAEAVPDHIENTEIKGRHDLRDE²⁵⁷ and Cys-⁵⁷⁶RKYLIEKSMDNKEVKRWEDK⁵⁹⁵, respectively) were custom synthesized and used for immunization in New Zealand white rabbits (Pacific Immunology). Horseradish peroxidase (HRP)-conjugated protein A (1/15,000 dilution) was from Cytiva (catalog number NA9120).

S. aureus cell pellets were homogenized with Lysing matrix B (MP Biomedicals, 100 mg beads/ml cells) in 25 mM Tris (pH 7.5) on a Retsch MM400 mixer mill at 15 Hz in four 3-min cycles. Clarified lysates were recovered by spinning at 20,817 × g at 4°C for 5 min to remove cell debris. A total of 0.1 to 0.2 A₂₈₀ unit of cell lysate were analyzed on 4% to 20% TGX SDS-PAGE gels (Bio-Rad), and the proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo system (Bio-Rad). The membrane was stained with Ponceau red (Amresco) to ensure equal loading, followed by immunoblotting using a 1:6,000 dilution of anti-HPF (39 (link)), a 1:1,000 dilution of anti-SigB (a gift from Markus Bischoff), and a 1:20,000 dilution of anti-CodY (ETU005; Kerafast). To detect multiple protein targets, the same membrane was stripped with the Restore Western blot stripping buffer (Thermo Fisher) and reprobed with the desired antibody. The intensity of immunoblot bands was quantitated by ImageJ.