Office scanner

Cell lines were lysed using RIPA buffer (Thermo-Fisher Scientific) supplemented with Halt^™ protease inhibitor (Thermo-Fisher Scientific). Cell debris was removed after centrifugation at 4°C, and total protein concentration was determined using the DC protein assay (Bio-Rad). Electrophoretic separation of protein (12 μg/well) was performed using 4-15% gradient polyacrylamide gels (Bio-Rad). Separated protein was transferred for 18 hours at 4°C onto PVDF membranes (Bio-rad). Membranes were blocked for one hour at room temperature in TBS containing 0.1% tween (TBS-T) with 5% fat-free milk, followed by overnight incubation at 4°C with mouse anti-human TfR1 antibody (Thermo-Fisher Scientific) (1:500 dilution) or mouse anti-human β-actin antibody (Cell Signaling Technology, Danvers, MA, USA) (1:10,000 dilution) in 5% fat-free milk with TBS-T. Membranes were washed in TBS-T and incubated for 30 minutes at room temperature with a 1:2000 dilution of horseradish peroxidase-conjugated rabbit anti-mouse antibody (Cell Signaling Technology) in 5% milk with TBS-T. Protein signals were developed on X-ray film using the Pierce ECL Western blotting substrate (Thermo-Fisher Scientific) or the SuperSignal West Femto Maximum Sensitive Substrate (Thermo-Fisher Scientific). X-ray film of blots was digitized using an office scanner (Epson, Long Beach, CA, USA).

For the cytoplasmic calcium mobilization assay [Fig. 4(a)], A. thaliana seedlings expressing the calcium reporter, aequorin,30 were grown on half‐strength Murashige–Skoog (½ MS) medium for 5 days. Single seedlings were incubated in 200 μL ½ MS media supplemented with 2.5 μM coelenterazine cp (Sigma‐Aldrich) in the dark for 16 h. RALF8 was added to the medium immediately before measuring bioluminescent emission. Luminescent signals were measured with a plate reader (Tecan) for 150 s. The total luminescence emission reflecting an increase in calcium concentration was calculated by summing signals obtained during 150 s. For a negative control, a volume of water or buffer equivalent to the RALF8 solution was added.
For the RALF8 root growth inhibition assay [Fig. 4(b,c)], wild‐type (Columbia ecotype) and fer‐4 knockout mutant seedlings were grown for 3 days on ½ MS solid medium and then transferred to 500 μL ½ MS liquid medium containing 0, 1, 2, or 5 μM RALF8 peptide. Seedlings were further grown for 2 days. Images were captured using an office scanner (Epson), and root length was quantified using ImageJ software.

After treatment, cells were trypsinized, counted, and 1000 or 5000 cells were plated in triplicate in 6-well plates containing complete RPMI medium and drug, as indicated. Cells were allowed to grow under standard conditions for at least 4 days until colonies were observed. For staining, 1 ml of crystal violet stain (0.05% crystal violet, 1% formalin, 1% methanol in PBS) was added to the cells. Cells were destained in deionized water, and images were taken using an Epson office scanner under film settings. To quantify stain, crystal violet was extracted using 1.5 ml of 1% SDS, followed by absorbance measurement at 612 nm wavelength light on a BioTek luminometer plate reader. Readings were also conducted in triplicate for each sample.