Journal: The Journal of Biological Chemistry

Article Title: Competition for cysteine acylation by C16:0 and C18:0 derived lipids is a global phenomenon in the proteome

doi: 10.1016/j.jbc.2023.105088

Figure Lengend Snippet: LAMTOR1 and HRAS as examples of differential S -acylation on multiple acylation sites. A , a scheme illustrating the N-terminal sequence of LAMTOR1 with its lipid modifications. B , APE assay detects acylation sites on the endogenous LAMTOR1 in HeLa and HEK293E cells. A representative image of three biological replicates. C , APE assay detects that both Cys3 and Cys4 are acylated in LAMTOR1-HA. HeLa cells were transiently transfected with overexpression constructs and lysed for APE assay 24 h post transfection. WT LAMTOR1 was compared to acylation mutants upon detection with anti-HA antibody (single mutants C3S and C4S and the double mutant C3S/C4S). The endogenous GNAI2 with its single acylation is shown as a positive control of the assay and tubulin as a loading control. A representative of three independent experiments. D , both Cys3 and Cys4 are acylated by either C15-az or C17-az in LAMTOR-HA. Metabolic labeling with azido fatty acids (conditions as in Fig. 1 A ) in HeLa cells was performed 24 h after transfection. The purified labeled proteins were eluted for a Western blot and the WT LAMTOR1 was compared to the acylation mutants (detected with anti-HA antibody). The efficiency of GNAI2 and TFR1 labeling serves as a positive control of the assay. A representative of two independent experiments. E , a scheme illustrating the C-terminal sequence of HRAS with its lipid modifications. F , APE assay fails to show acylation sites on the endogenous HRAS in HeLa and HEK293E cells, likely due to a faint antibody signal. A representative image of three biological replicates. G , both Cys181 and Cys184 are acylated in V5-HRAS, detected via APE assay. Transiently transfected HeLa cells were lysed for APE assay 24 h post transfection. WT HRAS was compared to acylation mutants upon detection with anti-V5 antibody (single mutants C181A and C184A and the double mutant C181A/C184A). The endogenous GNAI2 with its single acylation is shown as a positive control of the assay and tubulin as a loading control. A representative of three independent experiments. H , both Cys181 and Cys184 are acylated by both C15-az and C17-az in V5-HRAS. Metabolic labeling with azido fatty acids (conditions as in Fig. 1 A ) in HeLa cells was performed 24 h after transfection. The WT V5-HRAS was compared to the acylation mutants (detected with anti-V5 antibody). The efficiency of GNAI2 and TFR1 labeling serves as a positive control of the assay. A representative of three independent experiments. APE, acyl-PEG exchange; TFR, transferrin receptor.

Article Snippet: HEK293E and HeLa cells (CRL-10852 and CCL-2, respectively; ATCC), were cultivated in high-glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % fetal calf serum (Sigma-Aldrich), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Gibco) in a 37 °C incubator under stable 5 % CO 2 atmosphere.

Techniques: Sequencing, Transfection, Over Expression, Construct, Mutagenesis, Positive Control, Control, Labeling, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Competition for cysteine acylation by C16:0 and C18:0 derived lipids is a global phenomenon in the proteome

doi: 10.1016/j.jbc.2023.105088

Figure Lengend Snippet: Validation of preferential S -acylation of proteins with one fatty acid. A , metabolic labeling with azido fatty acids in HeLa cells was performed under the conditions described in Figure 1 A . After the click-based pulldown on alkyne agarose, labeled proteins were eluted and probed on Western blots. The efficiency of GNAI2 labeling serves as a positive control of the assay. B , the intensity of bands in A was quantified, the values were background corrected and normalized to the respective GNAI2 band. The ratio of the efficiency of C17-az and C15-az labeling is shown for each tested protein. The significance was tested by one sample t test (hypothetical mean 1), when significant, p -value is indicated, n = 2 to 5 (weak antibodies could not be detected in some samples), mean ± SD. C , metabolic labeling with azido fatty acids in HeLa cells was performed in the presence of increasing concentrations of the other unlabeled fatty acid. Cells were incubated with 100 μM C15-az and C17-az alone or together with 10, 100, and 1000 μM of C18:0 or C16:0, respectively. D , quantification of the competition experiments as shown in C .

Article Snippet: HEK293E and HeLa cells (CRL-10852 and CCL-2, respectively; ATCC), were cultivated in high-glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % fetal calf serum (Sigma-Aldrich), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Gibco) in a 37 °C incubator under stable 5 % CO 2 atmosphere.

Techniques: Biomarker Discovery, Labeling, Western Blot, Positive Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Competition for cysteine acylation by C16:0 and C18:0 derived lipids is a global phenomenon in the proteome

doi: 10.1016/j.jbc.2023.105088

Figure Lengend Snippet: TFR1 as an example of preferential S -acylation on multiple acylation sites. A , schematic diagram of amino acids 61 to 68 of TFR1, which contain lipid modifications. B , APE assay detects acylation sites on endogenous TFR1 in HeLa and HEK293E cells. C , both Cys62 and Cys67 are acylated in TFR1-HA, detected via APE assay. Transiently transfected HeLa cells were lysed for APE assay 24 h post transfection. WT TFR1 is compared to acylation mutants upon detection with anti-HA antibody (single mutants C62A and C67A, and the double mutant C62A/C67A). The endogenous GNAI2 with its single acylation is shown as a positive control of the assay and tubulin as a loading control. A representative of three independent experiments. D , both Cys62 and Cys67 are preferentially acylated with C15-az compared to C17-az. Metabolic labeling with azido fatty acids (conditions as in Fig. 1 A ) in HeLa cells was performed 24 h after transfection. The WT TFR1-HA was compared to the acylation mutants (detected with anti-HA antibody). The efficiency of GNA2 labeling serves as a positive control of the assay. A representative of two independent experiments. APE, acyl-PEG exchange; TFR, transferrin receptor.

Article Snippet: HEK293E and HeLa cells (CRL-10852 and CCL-2, respectively; ATCC), were cultivated in high-glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % fetal calf serum (Sigma-Aldrich), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Gibco) in a 37 °C incubator under stable 5 % CO 2 atmosphere.

Techniques: Transfection, Mutagenesis, Positive Control, Control, Labeling

Journal: PLoS ONE

Article Title: Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

doi: 10.1371/journal.pone.0024089

Figure Lengend Snippet: 293T cells were transfected with vectors expressing HA-R1 and one of Flag-R2 isoforms. At 45 h after transfection, protein extracts were subjected to immunoprecipitation (IP) with anti-HA or anti-Flag antibodies. IP materials and total cell lysates (TCL) were detected by Western blotting (WB) analysis. Data for R1 are representative of three IP samples.

Article Snippet: HeLa, HepG2 and 293T cells (ATCC Numbers: CCL-2, HB-8065, CRL-11268) were maintained in Dulbecco′s modified Eagle′s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 u/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL fungizone from Invitrogen, at 37°C in 5% CO 2 /air atmosphere.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Adeno-Associated Virus Capsids Displaying Immunoglobulin-Binding Domains Permit Antibody-Mediated Vector Retargeting to Specific Cell Surface Receptors

doi: 10.1128/JVI.76.9.4559-4566.2002

Figure Lengend Snippet: Retargeting to M-07e, Jurkat, and Mec1 cells by recombinant Z34C mutants and antibodies against CD29, CD117, and CXCR4 a

Article Snippet: Cell lines HeLa (ATCC CCL-2), 293 (ATCC CRL-1573), and Jurkat (ATCC CRL-1990) were provided by the American Type Culture Collection.

Techniques: Recombinant, Virus

Journal: Scientific Reports

Article Title: Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy

doi: 10.1038/s41598-017-14313-z

Figure Lengend Snippet: SMN protein is released from cells inside of extracellular vesicles. Panel A: Exosomes and microparticles were isolated from A549 cells, grown in medium supplemented with vesicle-depleted FBS, using differential centrifugation and their size profiles were determined using nanoparticle tracking analysis. Panel B: Protein from extracellular vesicles (5 μg) was subjected to SDS-PAGE and the resulting immunoblot was probed for Alix, TSG101, and SMN. Data is representative of n = 2. Panel C: Immunogold labeling of SMN protein was used to demonstrate the presence of SMN protein within A549-derived exosomes, scale bar represents 100 nm. Panel D: Higher magnification of electron microscopy images showing SMN protein contained in exosomes, scale bar represents 100 nm. Four representative images are shown.

Article Snippet: 293 , , A549 (ATCC CCL 185 , and HeLa (ATCC CCL 2 ) cells were grown in Minimum Essential Medium (MEM, Sigma, Oakville, ON) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM GlutaMAX (Invitrogen, Burlington, ON), and 1X antimycotic-antibiotic (Invitrogen, Burlington, ON).

Techniques: Isolation, Centrifugation, SDS Page, Western Blot, Labeling, Derivative Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Survival Motor Neuron Protein is Released from Cells in Exosomes: A Potential Biomarker for Spinal Muscular Atrophy

doi: 10.1038/s41598-017-14313-z

Figure Lengend Snippet: The quantity of SMN protein within exosomes reflects the level expressed within the cell from which they are derived. Panel A: A stable A549-based cell line that overexpresses a FLAG-tagged version of SMN protein was generated, designated A549::SMN cells, and the quantity of intracellular SMN protein was analysed by immunoblot using antibody to SMN and FLAG, relative to the parental A549 cell line. Equal protein loading was confirmed by probing the membrane with tubulin. Data is representative of n = 3. Panel B: Exosomes were isolated from medium of A549 or A549::SMN cells, 3 µg of the resulting samples were separated by SDS-PAGE on duplicate blots, and the resulting immunoblots were probed for SMN and Alix (loading control), or FLAG and Alix. Data is representative of n = 3. Panel C: A549 cells were infected at an MOI of 10 or 100 with AdSMN (or mock infected) and, 24 h post-infection, crude cellular protein lysates were prepared and analysed by immunoblot on duplicate blots for SMN and tubulin (loading control), or FLAG and tubulin. Data is representative of n = 3. Panel D: A549 cells were infected with an MOI 50 and media was collected for exosome purification. Equal volume of each sample was separated by SDS-PAGE and analysed by immunoblot for SMN and Alix (loading control). Data is representative of n = 3.

Article Snippet: 293 , , A549 (ATCC CCL 185 , and HeLa (ATCC CCL 2 ) cells were grown in Minimum Essential Medium (MEM, Sigma, Oakville, ON) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM GlutaMAX (Invitrogen, Burlington, ON), and 1X antimycotic-antibiotic (Invitrogen, Burlington, ON).

Techniques: Derivative Assay, Generated, Western Blot, Membrane, Isolation, SDS Page, Control, Infection, Purification

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Durability of transgene expression and vector integration: recombinant SV40-derived gene therapy vectors.

doi: 10.1006/mthe.2002.0657

Figure Lengend Snippet: FIG. 6. Southern blot analysis of genomic DNA from 293 cells treated with SV(BUGT). Human 293 cells were treated once with SV(BUGT), and their high-molecular weight DNA harvested 4days later. DNA from the transduced cells was restricted with NotI ± EcoRI, or was left uncut. These DNAs were elec- trophoresed, blotted, and hybridized to a 32P-labeled full- length SV(BUGT) DNA probe. Labeled molecular size markers are shown at left (HindIII digestion of phage DNA). An equal amount of unrestricted DNA from mock-trans- duced 293 cells was elec- trophoresed in the right lane.

Article Snippet: We treated HeLa and 293 cells (ATCC) separately using both approaches.

Techniques: Southern Blot, High Molecular Weight, Labeling