Anti phospho threonine antibody

Thylakoid proteins were extracted from the leaves in the presence of 10 mM NaF under dim light [59 (link)], and then were separated by SDS−PAGE (6% acrylamide stacking gel + 15% separation gel + 6 M urea) based on equal chlorophyll. Proteins from the gel were subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Immobilone, MilliPore, Darmstadt, Germany) using standard methods, and then were detected using specific antibodies including Lhca1, Lhca3, PsaD, CP43, D1, D2, Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5, and Lhcb6 (Agrisera, Umea, Sweden). Phosphorylation of thylakoid proteins was detected by anti−phosphothreonine antibody (Cell Signaling, Ipswich, MA, USA). The immunoblotting signals of proteins were detected using horseradish peroxidase−conjugated anti−rabbit antibody (Agrisera Comp., Umea, Sweden) and ECL reagents (GE Healthcare, Buckinghamshire, UK). Quantity one software (v4.4, Bio−Rad Comp., Hercules, CA, USA) was used to quantify signal amplitude.

One µg recombinant proteins (FemX, FemA or FemB) were incubated with 1 µg Stk kinase domain (Stk_KD) in kinase buffer (50 mM Tris-HCl pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂) with or without 20 mM ATP for 1 h at 37 °C. For the dephosphorlyation reaction, 1 µg Stp was added to the reaction mixture. Each reaction was stopped by addition of 5 x SDS sample buffer and heating for 5 min 95 °C. The phosphorylated proteins were detected by Western blotting using an anti-phosphothreonine antibody (#9381 S, Cell Signaling, Frankfurt, Germany). SDS-PAGE was performed and the proteins were blotted on a nitrocellulose membrane using a semi-dry blotting technique. The blot was blocked in 5% albumin (Sigma-Aldrich, Taufkirchen, Germany) in TBS (10 mM Tris-HCl pH 7.5, 0.9% NaCl) for 2 h and then incubated with anti-phosphothreonine antibody (1:5,000, 1% albumin/TBS-T) overnight at 4 °C. After washing the blot three times in TBS-T (TBS + 0.05% Tween 20) for 15 min, the blot was incubated with anti-rabbit-HRP antibody (#L3012, SAB, USA) (1:10,000 in TBS) for 2 h. After three additional TBS-T washing steps, the blot was incubated with ECL substrate for 2 min. Phosphorylated proteins were visualized using ImageQuant LAS 4000 imaging system (GE Healtcare, Munich, Germany).

M. tuberculosis H37Rv was used as the wild type and as the parental strain for all mutants. E. coli TOP10 and XL1 blue were used for cloning and were grown in LB broth supplemented with appropriate antibiotics (25 μg/ml kanamycin or 150 μg/ml hygromycin). M. tuberculosis H37Rv was grown at 37°C in Middlebrook 7H9 liquid medium (Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% NaCl, 0.2% Glycerol and 0.05% Tween 80 (7H9-ADN-Tw). Kanamycin (25 μg/ml) or hygromycin (50 μg/ml) was added to liquid or agar medium when appropriate. Restriction endonucleases and DNA modifying enzymes were purchased from New England Biolabs. Pristinamycin 1A (ptc) was purchased from Molcan Corporation (Canada). Anti-phosphothreonine antibody and Anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technology. Analytical grade chemicals and reagents were purchased from Sigma Aldrich. Details of primers, plasmids and strains are shown in S1 Table.

Thylakoid membrane proteins were isolated from Arabidopsis leaves with 10 mM NaF under dim light following the method of Chen et al. [65 (link)]. After measuring Chl concentrations from thylakoid membrane extracts, thylakoid proteins containing equal chlorophyll were separated by 15% SDS-PAGE with 6 M urea and then transferred to polyvinylidene difluoride (PVDF) membrane (Immobilone, Millipore, Darmstadt, Germany). Some PSI and PSII proteins were detected by specific antibodies against Lhca1-3, PsaD, CP43, D1, D2, Lhcb1-6, and PsbS, which were purchased from Agrisera (Umea, Sweden). In addition, phosphorylation of thylakoid membrane proteins was analyzed using an antiphosphothreonine antibody (Cell Signaling, Ipswich, MA, USA). For the detection of the immunoblots, horseradish peroxidase-conjugated secondary antibody (Agrisera, Umea, Sweden) and the ECL reagent (GE Healthcare Buckinghamshire, UK) were used. Quantification of the immunoblots was done using quantity one software (Bio-Rad Comp. Hercules, CA, USA).

Human myc-his-tagged XOR (pcDNA3.1mychisAhXOR) was expressed in 293A cells and purified using the HisPur cobalt purification kit (Thermo Scientific 90090) according to the manufacturer’s recommendations. Equal amounts of purified XOR protein was added to each reaction containing immune-precipitated CDK5, 1x kinase assay buffer, and Mg²⁺/ATP cocktail. Reactions were incubated at 30°C for 20 minutes and termination by addition of gel loading buffer and incubation at 95°C for 5 minutes. Each reaction was resolved on SDS-PAGE and probed with anti-phospho-threonine antibody (Cell Signaling, Boston, MA) and with an anti-myc antibody (Cell Signaling, Boston, MA).

Lysates were resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was incubated with the primary antibody followed by incubation with anti-rabbit, anti-mouse, or anti-goat immunoglobulin-G conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). Specific proteins were detected by using enhanced chemiluminescence (GE Healthcare, Backinghamshire, UK). The primary antibodies for Western blotting were as follows: anti-Notch1 antibody (Santa Cruz, Dallas, TX, USA), anti-Jagged1 antibody (Santa Cruz), anti-p53 antibody (DO-1) (Santa Cruz), anti-p21 antibody (Millipore, Billerica, MA, USA), anti-p16 antibody (BD Pharmingen, San Jose, CA, USA), anti-ID1 antibody (Santa Cruz), anti-phospho p38MAPK (Thr180/Tyr182) antibody (Cell signaling, Boston, MA, USA), anti-p38MAPK antibody (Cell signaling), anti-phospho SAPK/JNK (Thr183/Tyr185) antibody (Cell Signaling), anti-JNK1/3 antibody (Santa Cruz), anti-actin antibody (Cell signaling), anti-GAPDH antibody (Santa Cruz), anti-phosphoserine antibody (Abcam, Cambridge, UK) and anti-phosphothreonine antibody (Cell signaling). To assess the phosphorylation level of Id1, cell lysates were immunoprecipitated with FLAG M2 agarose (Sigma).