Starbright blue 700 goat anti rabbit igg

Samples were loaded into 12% stain-free Tris-glycine SDS-polyacrylamide gels prepared in house. Gels were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked at room temperature for 1 h in 5% milk. The following primary antibodies (Table S6) were diluted 1:1000 in 1% milk and incubated overnight: anti-myocilin (MAB3446, R&D Systems), anti-β-actin (4970, Cell Signaling Technology) and anti-FLAG (F3165, Sigma Millipore). After primary antibody incubation, PVDF membranes were washed three times with PBS containing 0.1% Tween-20. The following secondary antibodies were diluted 1:2500 in 1% milk and incubated on membranes for 1 h: Starbright Blue 520 goat anti-mouse IgG and Starbright blue 700 goat anti-rabbit IgG (Bio-Rad). Images were captured with a ChemiDoc MP imaging System (Bio-Rad). Quantification of western blots was done with ImageLab software (Bio-Rad).

Histone extracts were prepared with the EpiQuik Total Histone Extraction kit (Epigentek, OP-0006-100-EP; for MB, MT, and GAS) or the acid histone extraction protocol published by Abcam (mESC, ADIPO, BMDM, and PIM). Histone protein extracts were resolved using a gradient SDS-PAGE before being immunoblotted onto a PVDF membrane. The membrane was blocked for 1 h in blocking solution (TBS/0.1% Tween/5% BSA or milk) and then incubated overnight at 4°C with primary antibodies diluted in blocking solution. After washes with TBS/0.1% Tween, the membranes were incubated with secondary antibodies conjugated with fluorescent or HRP tag diluted in blocking buffer for 1 h at room temperature. The band signals were visualized using Bio-Rad ChemiDoc Imaging System. The following primary antibodies were used: H3 (Abcam, ab1791), pan-KLA (PTM Bio, PTM-1401), and H3K18la (PTM Bio, PTM-1406 or PTM-1406RM). The secondary antibodies used were an HRP-conjugated monoclonal donkey anti-rabbit IgG (1:5000, Amersham, NA934) and StarBright Blue 700 Goat Anti-Rabbit IgG (1:2500, Bio-Rad, 12004161)

Protein concentrations of whole-cell lysates and membrane fractions were measured using the BCA assay. Cells were resuspended in 20 mM Tris-HCl buffer (pH 8.0) and lysed with lysostaphin (40 μg mL⁻¹) in a 37°C heat block for 30 min. SDS loading buffer at 5× was added to the cell lysates, followed by heating for 5 min. Samples were subjected to 12% SDS-PAGE, and proteins were transferred to polyvinylidene difluoride PVDF membranes (Cytiva). Membranes were blocked in 5% (wt/vol) skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% [vol/vol] Tween 20, pH 7.6) for 1 h. Membranes were washed three times with TBST and incubated with rabbit polyclonal antibodies specific to SaeR (1:1,000 dilution) and SaeS (1:1,000 dilution) in blocking buffer for 1 h (20 (link)). Membranes were washed with TBST and incubated with StarBright Blue 700 goat anti-rabbit IgG (1:3,500; Bio-Rad) for 1 h. After a brief wash in TBST, the signals were visualized using an Amersham ImageQuant 800. The densities (mean intensity per unit area) of the SaeS and SaeR protein bands were determined by quantification with Multi Gauge software (Fujifilm).