Analysis of the possibility of UV-B and salt-induced aggregation of Pol λ <i>in vitro</i> and <i>in vivo</i>.

<p>A) Aggregation assay using untreated control purified recombinant full length and N terminus deletion mutants of Pol λ. Aggregation assay using (B) untreated control, UV-B irradiated and salt treated purified recombinant full length, (C) Del 2 and (D) Del 3 proteins, respectively. (E) UV-B mediated aggregation of purified bovine γ crystalline. (F) Aggregation of high salt treated chicken IgG. 0.1 mg/ml of purified untreated control, UV-B irradiated (~200 J/m² of UV-B treatment for 4 h at 25°C) or salt treated (500 mM NaCl treatment for 2 h at 25°C) protein samples in 50 mM Tris-HCl, pH 7.5 (with 1 mM PMSF and 1 mM β-mercaptothanol) were subjected to static light scattering at 360 nm. Absorbance was measured over a time course of 4 h at room temperature (25°C). Influence of UV-B irradiation and high salinity stress on Aggregation of Pol λ and its N terminus truncated forms <i>in vivo</i>. (G) and (H) Immunoblotting of protein extracts from soluble and insoluble (pellet) fractions of UV-B irradiated protoplasts from wild-type <i>Arabidopsis</i> (G) and tobacco leaf mesophyll cells transiently expressing AtPolλ (H). (I) and (J) Immunoblotting of protein extracts from soluble and insoluble (pellet) fractions from 7-days-old wild-type <i>Arabidopsis</i> seedlings (I) and <i>Agro</i>-infiltrated tobacco leaves (transiently expressing AtPolλ) exposed to increasing concentrations of NaCl (J). (K) and (L) Immunoblotting of protein extract from soluble and insoluble (pellet) fractions from UV-B irradiated protoplasts of transgenic <i>atpolλ-1</i> seedlings expressing Pol λ-Del-2 protein (<i>atpolλ-1</i>^{<i>Pol λ-Del 2</i>}) (K) or 7-days-old <i>atpolλ-1</i>^{<i>Pol λ-Del 2</i>} seedlings exposed to increasing concentrations of NaCl (L). (M) and (N) Immunoblotting of protein extracts from soluble and insoluble (pellet) fractions from UV-B irradiated protoplasts of transgenic <i>atpolλ-1</i> seedlings expressing Pol λ-Del 3 protein (<i>atpolλ-1</i>^{<i>Pol λ-Del 3</i>}) (M) or 7-days-old <i>atpolλ-1</i>^{<i>Pol λ-Del 2</i>} seedlings exposed to increasing concentrations of NaCl (N). Positions of DNA Pol λ in the soluble and pellet fractions have been indicated. Size markers are shown on the left of gel images. Ponceau S stained membranes with the soluble protein extracts showing RuBisCo large subunit (LSU) have been shown as loading controls (middle panel, Fig G-N). Representative data in Fig G-N are from three trials with similar results. Immunoblotting of protein extracts from <i>Arabidopsis</i> (Col-0 and transgenic lines) protoplasts or seedlings were carried out using affinity purified anti-AtPolλ polyclonal antibody (1:250 dilutions) while anti-c-Myc antibody (1:1000 dilutions) was used for immunodetection of DNA Pol λ in protein extracts from tobacco leaves or mesophyll cell protoplasts ectopically expressing AtPolλ. <i>atpolλ-1</i> is a null mutant line, devoid of AtPolλ expression. Representative gel images from at least three independent trials are shown.