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. 2021 Dec 4;22(23):13123. doi: 10.3390/ijms222313123

Figure 6.

Figure 6

Evidence for direct in vitro interaction of NtPhyt with calreticulin-3. Recombinant N. tabacum calreticulin-3 bearing an N terminal His6 tag (His-CRT3) was produced in Escherichia coli cells and immobilized on Ni-nitrilotriacetic acid (Ni-NTA) agarose (CRT3, approx. 0.5 μg protein per binding experiment). Ni-NTA agarose resin preincubated with an equivalent amount of lysed vector-only transformed E. coli cells was used as a control (control). Upon incubation of each resin with NtPhyt for 1 h, NtPhyt proteolytic activity in the flow-through fractions (a) and in the eluates from the column with EDTA-containing buffer (b) was fluorometrically determined using 20 μM Ac-VEID-AFC as a phytaspase substrate. Data represent the mean ± SD of three independent experiments. Significant differences to the control are shown as ** p < 0.01. The two-tailed p value is 0.0010 for (a) and 0.0011 for (b) (unpaired t-test).