Journal: Nature Communications

Article Title: A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences

doi: 10.1038/s41467-023-42746-w

Figure Lengend Snippet: Plasmids deposited at Addgene

Article Snippet: Primary antibodies used in this study: GFAP (1:1000, chicken, Rockland #200-901-D60); Aldh1l1 (1:400, rabbit, Abcam #Ab87117); Sox9 (1:1000, rabbit, Millipore Sigma #AB5535); NeuN (1:1000, chicken, Synaptic Systems #266 006); CD31 (1:100, rat, BD Biosciences #550274); V5 (1:400, human, Absolute Antibodies #AB00136-10.0); Myc (1:400, 488-labeled, Biotium #20436); FLAG (1:400, 543-labeled, Biotium #20433); HA (1:100, rat, Roche #11-867-423), and GFP (1:500, 488-labeled nanobody, ChromoTek #GBA488-100).

Techniques: Membrane, Plasmid Preparation, Expressing

Journal: Frontiers in Pharmacology

Article Title: Ectopic Overexpression of PPARγ2 in the Heart Determines Differences in Hypertrophic Cardiomyopathy After Treatment With Different Thiazolidinediones in a Mouse Model of Diabetes

doi: 10.3389/fphar.2021.683156

Figure Lengend Snippet: Overexpression of PPARγ2 is sufficient to lead to hypertrophy in cardiac myocytes. (A) Measurement of PPARγ2 mRNA levels in cardiomyocytes. (B) Analysis of protein levels with Western blot after overexpressing DDK-PPARγ2 in cardiomyocytes. (C) mRNA levels of the hypertrophic marker gene BNP after overexpressing PPARγ2 in cardiomyocytes. (D) representative images of α-SMA staining with CY3 (red) and nuclei with DAPI (blue) in cardiomyocytes. (E) quantified cell surface areas in cardiomyocytes. CON represents transfection of empty PCMV6 plasmid; OE represents transfection of the PCMV6-PPARγ2 plasmid. Means ± SEM for three independent experiments, * p < 0.05, ** p < 0.01 vs. CON.

Article Snippet: Membranes were blocked with skimmed milk powder (Bio-Rad, United States) for 1.5 h and incubated at 4°C overnight with primary antibodies against PPARγ2 (1:1,000, ab45036, Abcam), DDK (1:2,000, TA50011, OriGene), and GAPDH (1:500, TA505454, ZSGB-Bio).

Techniques: Over Expression, Western Blot, Marker, Staining, Transfection, Plasmid Preparation

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: bioRxiv

Article Title: Minimal genetically encoded tags for fluorescent protein labeling in living neurons

doi: 10.1101/2021.01.14.426692

Figure Lengend Snippet: a , A schematic representation of genetic code expansion and labeling of proteins by click chemistry. Cells are transfected with plasmids bearing genes that encode for NES PylRS (a pyrrolysyl-tRNA synthetase with a nuclear export signal) and tRNA CUA Pyl , as well as a TAG stop codon-containing gene that encodes for the protein of interest (POI). The NES PylRS charges tRNA CUA Pyl with the unnatural amino acid (UAA), this charged tRNA CUA Pyl recognizes the UAG stop codon in the mRNA encoding the POI, and UAA is co-translationally incorporated into the POI. In the subsequent step, a tetrazine derivative of a fluorescent dye reacts with the UAA by strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reaction, and the dye is attached directly to the POI. b , Chemical structures of UAAs and tetrazine dyes that were used to obtain the data shown in the main figures. c , ND7/23 cells expressing NFM, NES PylRS/tRNA CUA Pyl , and wild-type NFL WT -FLAG, stained with an anti-FLAG antibody, followed by Alexa Fluor (AF) 488-conjugated secondary antibody. d , ND7/23 cells expressing NFL K363TAG -FLAG, neurofilament medium chain (NFM), and NES PylRS/tRNA CUA Pyl . Cells were incubated overnight with TCO‪A-Lys and then click labeled with silicon rhodamine-tetrazine (SiR-tz). Afterwards, cells were fixed and stained with the anti-FLAG antibody, followed by AF488-conjugated secondary antibody. Z -stack images were acquired with a confocal scanning microscope, and are shown as maximum intensity projections. Scale bars: 20 µm ( c , d ).

Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-FLAG antibody (Merck Millipore cat. no. F7425), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore cat. no. MAB1615, goat anti-rabbit Alexa Fluor (AF) 488 Plus (Thermo Fisher Scientific, cat. no. A32731), goat anti-rabbit AF555 (Thermo Fisher Scientific, cat. no. A21429), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), and goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728).

Techniques: Labeling, Transfection, Expressing, Staining, Incubation, Microscopy

Journal: bioRxiv

Article Title: Minimal genetically encoded tags for fluorescent protein labeling in living neurons

doi: 10.1101/2021.01.14.426692

Figure Lengend Snippet: a , b , MCNs expressing NFL K363TAG -FLAG, NFM and NES PylRS/tRNA CUA Pyl in the presence of TCO‪A-Lys. a , Three days after transfection, neurons were labeled with SiR-tz via click chemistry, then fixed and stained with anti-FLAG antibody, followed by AF488-conjugated secondary antibody. After fixation and immunocytochemistry labeling, neurons were imaged on a confocal scanning microscope. b , Three days after transfection, neurons were labeled with either BODIPY-tz, TAMRA-tz, or SiR-tz. Excess dye was washed for 3 h, and live neurons were imaged on a confocal scanning microscope. In TAMRA-tz panel, background staining of lysosomes is highlighted in both NFL K363TAG -expressing neuron (arrows) and in non-transfected neurons (arrowheads). Z -stack images are shown as maximum intensity projections. Scale bars: 20 µm ( a , b ).

Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-FLAG antibody (Merck Millipore cat. no. F7425), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore cat. no. MAB1615, goat anti-rabbit Alexa Fluor (AF) 488 Plus (Thermo Fisher Scientific, cat. no. A32731), goat anti-rabbit AF555 (Thermo Fisher Scientific, cat. no. A21429), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), and goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728).

Techniques: Expressing, Transfection, Labeling, Staining, Immunocytochemistry, Microscopy

Journal: bioRxiv

Article Title: Minimal genetically encoded tags for fluorescent protein labeling in living neurons

doi: 10.1101/2021.01.14.426692

Figure Lengend Snippet: a , A schematic representation of the experimental workflow. Eight days after plating, MCNs were transfected with NFL K363TAG -FLAG, NFM, and NES PylRS/tRNA CUA Pyl constructs. After 2 days of incubation with TCO‪A-Lys, neurons were labeled with the first dye (BODIPY-tz), incubated with TCO‪A-Lys for a further 2 days, and labeled with the second dye (SiR-tz). After the second labeling step, neurons were either fixed, stained with anti-FLAG antibody followed by AF555-conjugated secondary antibody, and imaged on a confocal scanning microscope ( b ), or live neurons were imaged on a confocal scanning microscope ( c ). Z- stack images are shown as maximum intensity projections. Scale bars: 20 µm ( b , c ).

Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-FLAG antibody (Merck Millipore cat. no. F7425), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore cat. no. MAB1615, goat anti-rabbit Alexa Fluor (AF) 488 Plus (Thermo Fisher Scientific, cat. no. A32731), goat anti-rabbit AF555 (Thermo Fisher Scientific, cat. no. A21429), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), and goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728).

Techniques: Transfection, Construct, Incubation, Labeling, Staining, Microscopy

Journal: bioRxiv

Article Title: Minimal genetically encoded tags for fluorescent protein labeling in living neurons

doi: 10.1101/2021.01.14.426692

Figure Lengend Snippet: MCNs expressing NFL K363TAG -FLAG, NFM and NES PylRS/tRNA CUA Pyl in the presence of TCO‪A-Lys. a – c , Two to three days after transfection, neurons were labeled with SiR-tz, fixed and stained with anti-FLAG antibody. Afterwards, neurons were imaged with STED super-resolution microscopy. Panel a shows a maximum projection of a confocal Z -stack of the neuron. The next four images show the confocal ( b ) and STED ( c ) micrographs of the region within the dashed box before and after deconvolution. d , The increase in resolution is visualized in a graphical representation of signal intensities across the line profiles drawn in the deconvolved confocal (orange line) and STED (blue line) images. e , STED imaging of two populations of click-labeled NFL. Two days after transfection, neurons were labeled with SiR-tz and incubated for a further 2 days with TCO‪A-Lys. Afterwards, cells were fixed, labeled with ATTO488-tz and imaged with STED super-resolution microscopy. Raw confocal and STED images were deconvolved using Huygens deconvolution software. Scale bars: 5 µm ( a – c, e ).

Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-FLAG antibody (Merck Millipore cat. no. F7425), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore cat. no. MAB1615, goat anti-rabbit Alexa Fluor (AF) 488 Plus (Thermo Fisher Scientific, cat. no. A32731), goat anti-rabbit AF555 (Thermo Fisher Scientific, cat. no. A21429), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), and goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728).

Techniques: Expressing, Transfection, Labeling, Staining, Microscopy, Imaging, Incubation, Software

Journal: bioRxiv

Article Title: Minimal genetically encoded tags for fluorescent protein labeling in living neurons

doi: 10.1101/2021.01.14.426692

Figure Lengend Snippet: a , A schematic representation of the tagging of endogenous NFL. MCNs were transfected with a pORANGE plasmid encoding for spCas9 and gRNA, and containing linker-3xFLAG or linker A6TAG -3xFLAG donor sequence. SpCas9 and gRNA are expressed from the plasmid, cut target sequences around the donor sequence and at the end of the Nefl gene. The resulting donor sequences are used by the non-homologous end joining (NHEJ) system to repair a double strand break in the genomic DNA. Endogenous NFL bearing the linker-3xFLAG sequence is transcribed and translated with the addition of the linker-3xFLAG tag at the C terminus. Endogenous NFL bearing the linker A6TAG -3xFLAG tag is fully transcribed. In the absence of a UAA, translation finishes at the UAG codon and full-length NFL is synthesized. In the presence of a UAA and NES PylRS/tRNA CUA Pyl , the UAA is incorporated in response to the UAG codon, the NFL-linker-UAA-3xFLAG is synthesized, and can be click labeled with a tetrazine dye. b , MCNs transfected with pORANGE NFL linker-3xFLAG knock-in construct, stained with anti-FLAG and anti-NFL antibodies, after 6 days of expression. The anti-FLAG and anti-NFL antibodies were stained with AF488- and AF647-conjugated secondary antibodies, respectively. c , MCNs transfected with pORANGE NFL linker A6TAG -3xFLAG knock-in, NES PylRS/tRNA CUA Pyl and eukaryotic release factor 1 mutant E55D (eRF1 E55D ) constructs. After incubation with TCO‪A-Lys for 6 days, the endogenous NFL-linker-UAA-3xFLAG was labeled with SiR-tz, then neurons were fixed and stained with anti-FLAG antibody followed by AF488-conjugated secondary antibody. Scale bars: 20 µm ( b , c ).

Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-FLAG antibody (Merck Millipore cat. no. F7425), mouse anti-neurofilament 70 kDa antibody, clone DA2 (Merck Millipore cat. no. MAB1615, goat anti-rabbit Alexa Fluor (AF) 488 Plus (Thermo Fisher Scientific, cat. no. A32731), goat anti-rabbit AF555 (Thermo Fisher Scientific, cat. no. A21429), goat anti-rabbit AF647 Plus (Thermo Fisher Scientific, cat. no. A32733), and goat anti-mouse AF647 Plus (Thermo Fisher Scientific, cat. no. A32728).

Techniques: Transfection, Plasmid Preparation, Sequencing, Non-Homologous End Joining, Synthesized, Labeling, Knock-In, Construct, Staining, Expressing, Mutagenesis, Incubation