Journal: bioRxiv

Article Title: SNF1-related protein kinase 2 directly regulate group C Raf-like protein kinases in abscisic acid signaling

doi: 10.1101/2020.02.04.933978

Figure Lengend Snippet: (A) AlphaScreen ® assay shows interaction of Raf36 and subclass III SnRK2s. Bars indicate means ± standard error (n=3), and asterisks indicate significant differences by Student’s t test ( P < 0.05). (B) Yeast two-hybrid (Y2H) assay shows interaction between Raf36 and subclass III SnRK2s. Yeast cells expressing GAL4AD:Raf36 and GAL4BD:SnRK2s fusion proteins were incubated on SD media supplemented with or without 3-amino-1,2,4-triazole (3-AT) and lacking combinations of amino acids leucine (L), tryptophan (W) and histidine (H), as follows (in order from low to high stringency): −LW, −LWH, −LWH +10 mM 3-AT, −LWH +50 mM 3-AT. Photographs were taken at 10 days (SRK2D and SRK2E) or 12 days (SRK2I) after incubation. (C) Subcellular localization of Raf36-GFP in leaf mesophyll cells. Chl indicates chlorophyll autofluorescence. Scale bar, 20 µm. (D) BiFC assays for Raf36 and subclass III SnRK2s. SnRK2 and Raf36 were transiently expressed in N. benthamiana leaves by Agrobacterium infiltration. Empty vector constructs were used as negative controls. nEYFP and cEYFP represent the N- and C-terminal fragments of the EYFP, respectively. BF indicates bright field images. Scale bar, 50 µm. (E) Y2H assay for truncated versions of Raf36 and SRK2E. Yeast cells co-expressing GAL4AD:Raf36, Raf36 N or Raf36 KD+C and GAL4BD:SRK2E fusion proteins were incubated on SD media lacking L, W, H, and adenine (A), as follows (in order from low to high stringency): −LW, −LWH, −LWHA.

Article Snippet: The AlphaScreen ® (Amplified Luminescent Proximity Homogeneous Assay) was carried out using an AlphaScreen ® FLAG ® (M2) Detection Kit (Perkin Elmer, MA) to detect protein-protein interactions.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Y2H Assay, Expressing, Incubation, Plasmid Preparation, Construct

Journal: Journal of cell science

Article Title: Improperly folded green fluorescent protein is secreted via a non-classical pathway.

doi: 10.1242/jcs.00047

Figure Lengend Snippet: Fig. 1. GFP is secreted via a non-classical pathway in CHO cells. (A) Untransfected CHO cells (Ctrl) and cells transiently transfected with either GFP (GFP) or preprolactin-myc (PPL) were labeled with [35S]-methionine and chased for 6 hours. The cell lysates (C) and media (M) were subjected to immunoprecipitation with either antibodies against GFP (lanes 1-8) or myc (lanes 9-12), and the washed immunoprecipitates were displayed by SDS-PAGE and fluorography. Some cells (+BFA) were treated with 1 µg/ml brefeldin A for 1 hour prior to labeling (lanes 3- 4, 7-8 and 11-12). ‘% Sec.’ denotes the percentage of secretion of GFP into the media as determined by a phosphorimager. (B) CHO cells transfected with plasmids encoding either mouse dihydrofolate reductase (mDHFR) or Schistosoma japonicum glutathione S-transferase (GST) both tagged with the FLAG epitope. The cells were labeled with [35S]-methionine and chased in serum-free medium. The cell lysate (C) and media (M) were immunoprecipitated with anti-FLAG antibodies and processed as above. (C) Plasmids coding for mDHFR and GFP were used to co-transfect CHO cells. The cells were labeled, chased and processed as described in B.

Article Snippet: Polyclonal antibodies against the FLAG epitope (DYKDDDDK) were purchased from ProSci.

Techniques: Transfection, Labeling, Immunoprecipitation, SDS Page, FLAG-tag

Journal: PLoS ONE

Article Title: Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

doi: 10.1371/journal.pone.0141618

Figure Lengend Snippet: Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.

Article Snippet: This involved incubating cell lysates with the Anti-FLAG M2 beads in Eppendorf tubes at 4°C overnight.

Techniques: Transfection