UGPase | UDP-glucose pyrophosphorylase (cytoplasm marker)

RVE8 protein accumulates in the subjective afternoon.Col, RVE8::RVE8-HA and 35S::RVE8-HA seedlings were entrained in 12 hr light:12 hr dark for 6 days before being released to constant white light (55 mol m−2 s−1) (A) Plants were harvested at the indicated times and extracts were subjected to immunoblot analysis using either an anti-HA antibody (upper panel) or an anti-UGPase antibody (lower panel). (B) Data shown in panel (A) was quantified using ImageQuant software. Data are representative of two independent experiments.

AtTSPO accumulation and chloroplast localization upon salt stress. (A) Immunoblot analysis of OxAtTSPO:eGFP (OxM1TSPO:eGFP, OxM21TSPO:eGFP and OxM42TSPO:eGFP) fusion proteins detected in plants with an antibody to GFP. Plants were untreated, or treated with 150 mM NaCl. As a control wild-type plants and plants over-expressing GFP (OxeGFP) seedlings were used. (B) Anti-GFP immunoblot of trypsinized chloroplasts from Arabidopsis plants either untreated or treated with 150 mM NaCl. Control immunoblots were probed with antibodies chloroplast proteins RuBisCo and D1; and to cytosolic UGPase. Each lane represents equal amounts of chloroplasts.

A simple workflow combining nuclear enrichment with quantitative mass spectrometry allows the study of host nuclear processes during infection. (a) Method overview. Detached leaves from 4‐wk‐old tomato plants were spray‐inoculated with Phytophthora capsici zoospores at a concentration of 500 000 spores ml−1, or water as a control (i). Leaves were harvested 8 and 24 h post‐infection and subject to nuclear enrichment (ii). Nuclear protein extracts were generated and fractionated in‐gel and digested with trypsin (iii). Peptide samples were subjected to liquid chromatography−tandem mass spectrometry (LC‐MSMS) analysis and peptide identification and label‐free quantification was carried out using the maxquant and perseus software packages, to identify proteins that were differentially expressed during infection. Nuclear predictions were performed using prediction software on our in‐house Galaxy server as described in the Methods. Subsequent data filtering was completed using the r software package (iv and v). Three independent biological replicates were generated. I, infected samples; NI, noninfected samples (v). (b) Successful enrichment of nuclear proteins demonstrated by western blotting with anti‐histone H3 antibody and subcellular markers. Protein extracts from enriched nuclear samples were compared to a total protein extract (TP) from tomato leaves. Protein concentrations were adjusted for equal loading. Nonnuclear contamination was assessed by probing with anti‐UDP‐glucose pyrophosphorylase (UGPase) antibody (cytoplasm) and calnexin homologue 1/2 antibody (endoplasmic reticulum). Samples were also run on gels and stained with Coomassie brilliant blue (CBB) to assess protein loading and Rubisco abundance. The figure shows the results from a single biological replicate. Blots for all three replicates are provided in Supporting Information Fig. S2.

RGL2, GAI, RGA and ABI5 antibodies specificity.Protein gel blot analysis using antibodies to RGL2 (A), ABI5 (B), GAI and RGA (C) as indicated. Protein extracts from Col-0 (WT), rgl2-13 (rgl2), rgl2-SK54 rga-28 (rgl2 rga), rgl2-SK54 rga-28 gai-t6 (rgl2 rga gai) and abi5-3 (abi5) mutant seeds harvested after seed imbibition in presence or absence of PAC (PAC) as indicated. UGPase protein levels are used as a loading control.

AMB and PAC induce different changes on DELLA protein accumulation.AMB promotes DELLA factor activity to stimulate ABA signaling. (A-C) Protein gel blot analysis using antibodies to RGL2 (A), RGA (B) and GAI (C) as indicated. Protein extracts from WT (Col seeds harvested 24 hr after seed imbibition in absence (MS) or presence of 10 µM PAC (PAC), 5 µM ABA (ABA) or 50 µM AMB (AMB). UGPase protein levels are used as a loading control. (D) AMB does not interfere with GA-dependent RGL2 protein downregulation. WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high RGL2 accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGL2 protein levels are followed by protein gel blot analysis over the time points indicated in red. UGPase protein levels are used as a loading control. Germination percentage at each time point is indicated. The asterisk (*) represents and unspecific banc detected by the RGL2 antibody. (E) AMB stimulates DELLA activity to promote ABA-dependent responses. Histograms show RGL2, ABI5, EM1 and NCED6 mRNA accumulation in WT (Col) and Δdella seeds treated as described in A. For each time point, mRNA levels are normalized to mRNA levels in WT seeds sown in absence of AMB. Data represent mean ±standard deviation (three replicates).AMB does not interfere with GA-dependent GAI and RGA protein downregulation.WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high DELLA accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGA (A) and GAI (B) protein levels are followed by protein gel blot analysis over the time points indicated in red as indicated. UGPase protein levels are used as a loading control.