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Once a bilayer was formed, the membrane or soluble fractions containing the activated Cry1Ab toxins (wild type, mutant or mixture of wild type with mutant) were added to the cis compartment.

WT Rpn12250 and each mutant were added in 5-fold excess to N-labelled Rpn10.

After drying, 50 µl drops containing 10 cells of wild-type and Δ yfh1 mutant were added and plates were incubated for 2 3 days at 30°C.

The plate was rinsed three times with PBS and 1×10 of the C. jejuni wild-type strain and the flpA mutant were added to each well and incubated for 1 h at 37 °C.

For the GST pull-down assays shown in Figure 4E, 12 g of purified GST, GST-TNRC6B SD or the corresponding ΔPAM2 mutant were added to lysates from E. coli cells expressing MBP-tagged HsPABPC1, MBP-DmPABPC1 or the corresponding mutants lacking the MLLE domain in a total volume of 1 ml of binding buffer (10 mM Hepes (pH 7.5), 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA and 1% [v/v] Triton-X100).

Critically, osteoblasts showed signs of both phosphate (Fig 5A) and calcium deposition (Fig 5B) when S. aureus SpA mutant was added.

For the T50 measurements, 125 μl of clarified lysate from a single mutant was added to all 12 wells in a row of a 96-well hard-shell thin-wall microplate (MJ Research).

For the [urea]50 measurements, 125 μl of the clarified lysate from a single mutant was added to all 12 wells in a row of a 96-well microtiter plate.

Three-fold serial dilutions of Con-anc or its mutants were added to the confluent cells.

Arrestin mutants were added at concentrations of both 2 and 5 μM, and the fluorescence was monitored over time.

IGFBP-2 or mutants were added to test their ability to inhibit IGF-I rescue of cells from butyrate-induced apoptosis.