transient transfection hela  (ATCC)

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 1 Flotillin-2 binds EFR3A in DRMs of HeLa cells. DRM fraction from HeLa cells diluted 1:1 with 2% octyl glucoside in 40 mM Tris–HCl, pH 7.4, 300 mM NaCl and protease inhibitor cocktail (Sigma–Aldrich) were incubated overnight with flotillin-2 Sepharose resin at 4 °C. Then the resin was thoroughly washed with the same buffer and suspended in sample buffer (20% SDS, 50% glycerol, 25 mM EDTA, 250 mM DTT, and 0.25 M Tris pH 6.8), boiled for 5 min, and subjected to SDS-PAGE. A Example of sequence coverage for EFR3A resulting from MS/MS identification of proteins bound to flotillin-2 Sepharose. SDS-PAGE gel fragment containing protein bands larger than 60 kDa was cut out and subjected to MS/MS identification (see also Additional file 1: Figure S1 and Table S1). B Western blotting of samples derived from a pull-down assay. Only bound fraction and unconjugated resin control are shown. Nitrocellulose was probed with an EFR3A antibody (Abnova). C Co-IP results, whole cell lysates of HeLa cells were incubated with 5 μg of anti-FL2 goat antibodies (Abcam) to protein G Dynabeads (Life Technologies) overnight at 4 °C with rotation, according to the manufacturer’s protocol. Nonimmune rabbit IgG (5 μg) was used as a negative control. Input means whole cell lysate proteins, 20 μg protein/well. Immunoprecipitated proteins were eluted in a sample buffer and analyzed by western blotting with rabbit anti-EFR3A antibodies and goat anti-flotillin-1 antibody as a positive control

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Protease Inhibitor, Incubation, SDS Page, Sequencing, Tandem Mass Spectroscopy, Western Blot, Derivative Assay, Pull Down Assay, Control, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Positive Control

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 2 Bacterially expressed recombinant EFR3A binds to recombinant flotillin-2 in vitro, in the overlay assay. Recombinant EFR3A protein was purified on metal ion chelating resin (Ni2+) and subjected to SDS-PAGE followed by transfer onto nitrocellulose. Then, membrane strips containing bands corresponding to molecular weight larger than 70 kDa were incubated with increasing concentrations of recombinant flotillin-2, followed by incubation with goat anti-flotillin-2 antibodies (Abcam) and secondary donkey anti-goat antibodies (Santa Cruz)

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Recombinant, In Vitro, Overlay Assay, Purification, SDS Page, Membrane, Molecular Weight, Incubation

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 3 EFR3A is present in the DRM fraction and its localization in the DRM fraction is sensitive to the removal of cholesterol from the HeLa cell plasma membrane. A Western blotting of sucrose step gradient fractions obtained after ultracentrifugation of cold 1% Triton X-100 extract (30 min on ice) from HeLa cells. 25 × 106 cells were used. Then, fractions were collected and proteins were precipitated with 10% TCA and analyzed with appropriate primary and secondary antibodies. B Western blotting of the Triton extract from cells treated with 8 mM β-methyl-cyclodextrin shows that localization of EFR3A in the DRM fraction is sensitive to the removal of cholesterol; for comparison, anti-EGFR, and anti-flotillin antibodies reactions are shown. For total protein and cholesterol profiles see Additional file 1: Fig. S3

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Clinical Proteomics, Membrane, Western Blot, Comparison

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 4 Silencing expression of the EFR3A gene in the Hela cell line induces changes in lateral membrane organization. Stable cell lines were obtained using EFR3A shRNA lentiviral particles or “scrambled” shRNA lentiviral particles as described in the Methods section. A Cell extracts of control or transduced cell lines were submitted to SDS-PAGE and western blotting probed with anti-EFR3A antibodies. GAPDH visualization was used as a loading control. B Quantitation of the EFR3A fractions shown in A. C Marked decrease of EFR3A level in cells is accompanied by a substantial reduction of EGFR in the DRM fraction. D Examples of FLIM images of the cell plasma membrane and E GPMVs derived from EFR3A KnD and “scrambled” cells. F and G Quantitation of FLIM data of EFR3A KnD and “scrambled” cells and corresponding GPMVs. For FLIM measurements of the fluorescence lifetime of the membrane-order sensitive probe, di-4 ANEPPDHQ (Invitrogen) control “scrambled” and EFR3A KnD HeLa cells were grown in LabTek chambers in DMEM medium with 10% FBS. After 24 h cells were washed twice and stained with a 2 μM di-4 probe in DMEM medium for 5 min. Cells were washed twice in HBSS buffer with 10 mM HEPES pH 7.4 and measurements were performed. Giant plasma membrane vesicles (GPMV) were generated from EFR3A KnD and “scrambled” cells as mentioned in the Methods section and stained with di-4 (2 μM) as above. The lifetime values of wild-type (WT) cells did not differ from “scrambled”, so we did not include them for clarity

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Membrane, Stable Transfection, shRNA, Control, SDS Page, Western Blot, Quantitation Assay, Clinical Proteomics, Derivative Assay, Fluorescence, Staining, Generated

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 5 “Rescue expression” of EFR3A in KnD HeLa cells. A Western blotting of total protein extracts of EFR3A KnD HeLa cells transfected with p3xEFLAG-CMV10 EFR3A (lane 1) and control p3xEFLAG-CMV10 plasmids (lane 2). Anti-FLAG antibodies (Merck) were used as primary antibodies. B Representative di-4 FLIM images of control, “scrambled” cell (left), EFR3A KnD cell transfected with an empty vector (middle), and “rescue transfected” (right). C Examples of di4 FLIM images of GPMVs generated from control, “scrambled” cells (left), EFR3A KnD cells transfected with an “empty” vector (middle), and EFR3A KnD cells transfected with a “rescue” plasmid p3xEFLAG-CMV10 EFR3A. D and E Quantitative distribution of fluorescence lifetime values obtained from FLIM analyses of di-4 labeled cells. D Plasma membrane and GPMVs. E FLIM analyses presented here were performed 48 h following transfection. F Dot-blot analysis of GPMVs derived from “rescue”-transfected EFR3A KnD cells using an anti-FLAG antibody. GPMVs were induced using a buffer containing NEM and CaCl2 48 h after transfection. Then, from the collected vesicles, proteins were precipitated with 10% TCA, washed, and resuspended in a reducing buffer containing DTT. Samples were applied on nitrocellulose using a dot blotter and probed with anti-FLAG antibodies

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Control, Plasmid Preparation, Generated, Fluorescence, Labeling, Clinical Proteomics, Membrane, Dot Blot, Derivative Assay

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 6 Mobility of the raft probe in EFR3A KnD partially returns towards normal in “rescue”-transfected EFR3A KnD HeLa cells svFCS measurements. A Diffusion time as a function of waist surface area. Black line: control HeLa “scrambled”, red line: EFR3A KnD, and green line: “rescue”-transfected EFR3A KnD HeLa cells. Cells were grown in LabTek chambers in DMEM medium with 10% FBS. After 24 h cells were washed three times and stained with 0.075 μM BODIPY-SM in HBSS with 10 mM HEPES and 0.075 μM BSA medium for 10 min. Cells were washed three times and measurements were performed in HBSS buffer with 10 mM HEPES, pH 7.4. For comparison, wild-type cells were treated with 8 mM β-methyl cyclodextrin for 1 h at 37 °C B. T0 and apparent diffusion coefficient. SEM, standard error of the mean

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Transfection, Diffusion-based Assay, Control, Staining, Comparison

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 7 Silencing of EFR3A gene expression affects G1 and G2/M length and proliferation but not cell mobility. A Cell cycle phase distribution in HeLa cells was evaluated via PI staining followed by flow cytometry presented as histograms. B Quantitation of flow cytometry analysis. Error bars represent the SEM of three independent experiments. *p < 0.05. The percentage of G1 phase cells increased within 48 h to 56%. C Cell proliferation was evaluated by WST-1 staining according to the Methods section. **Indicates a p ≤ 0.01 compared with control cells. D Representative images from the wound healing assay showing changes in HeLa cells with decreased EFR3A gene expression in comparison with control “scrambled” cells under stimulation with EGFR (50 ng/ml) for 48 h. E Quantitative analysis from the wound healing assay in the form of a bar graph showing the ratio of wound closure in EFR3A KnD versus “scrambled” cells. The observed reduced motility in cells with decreased expression of EFR3A in comparison with “scrambled” cells was found to be statistically insignificant (p-value = 0.158)

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Gene Expression, Staining, Flow Cytometry, Quantitation Assay, Control, Wound Healing Assay, Comparison, Expressing

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 8 Silencing of EFR3 gene expression affects PLCγ1 and EGFR phosphorylation. A Effect of EFR3A gene silencing on activation of EGF-induced EGF receptor and phospholipase PLCγ1 in HeLa cells via western blotting analysis. B Effect of MβCD on EGFR and PLCγ1 phosphorylation upon EGF treatment of WT HeLa cells. Fifteen micrograms of cell lysates was loaded on the 10% SDS-PAGE gel. C and D Quantitative analysis of data presented in A as bar graphs of fold change calculated as the ratio of relative levels of phospho-PLCγ and pEGFR normalized to the corresponding PLCγ1 and EGFR signal respectively. Significance values were calculated by paired Student’s t-test for **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Gene Expression, Phospho-proteomics, Activation Assay, Western Blot, SDS Page

Journal: Cellular & molecular biology letters

Article Title: EFR3A: a new raft domain organizing protein?

doi: 10.1186/s11658-023-00497-y

Figure Lengend Snippet: Fig. 10 Proposed model of EFR3A participation of raft domain regulation. Flotillin (most likely flotillin-1/-2 heterodimer) is a member of preexisting unstable protein–lipid complexes (τ1/2< 0.1 ms; < 10 nm in diameter) composed of few proteins and lipids which upon interaction with a protein–raft domain organizer, such as EFR3A, cluster into raft domains [resting state rafts, i.e., domains ~ 20 nm in diameter, which are considered more stable (τ1/2 ~ 1 s) and functional]. Protein clustering modulates membrane bilayer physical properties, such as fluidity and local diffusion

Article Snippet: shRNA lentiviral particle’s transduction and transient transfections HeLa EFR3A knockdown (KnD) and HeLa “scramble” were performed using EFR3A shRNA lentiviral particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, TX, USA) according to the manufacturer’s protocol.

Techniques: Functional Assay, Membrane, Diffusion-based Assay

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: Knockout of MED1 decreases the nuclear population of wild-type (WT) TRα1. mCherry tagged WT TRα1 (A), P398R TRα1 (B), C392X TRα1 (C), and A263V TRα1 (D) expression plasmids were transfected into MED1+/+ and MED1−/− MEFs. Cells were analyzed by fluorescence microscopy 24 hours post-transfection. Representative images are shown. Scale bar = 10 μm. (E) Bars represent the average normalized N/C ratios, calculated by normalizing the mutant N/C ratio to WT (MED1+/+). Error bars indicate +/− SEM (n=3 biologically independent replicates, with 60 cells per replicate). *** p<0.001, ns p>0.05.

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques: Knock-Out, Expressing, Transfection, Fluorescence, Microscopy, Mutagenesis

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: MED1 overexpression increases the N/C ratio of P398R TRα1 at elevated T3 levels. mCherry-tagged WT TRα1 (A), P398RX TRα1 (C), and C392X TRα1 (E) expression plasmids were either single transfected or cotransfected with GFP-MED1 into HeLa cells at physiological, 0 nM, or 100 nM T3 levels. Cells were analyzed by fluorescence microscopy 24 hours post-transfection. Representative images are shown. Scale bars = 10 μm. Average normalized N/C ratios were calculated by normalizing the mutant N/C ratio to WT. Bars represent the average normalized N/C ratio of WT TRα1 (B), P398R TRα1 (D), or C392X TRα1 (F) under different T3 levels. Error bars indicate +/− SEM (n=3 biologically independent replicates, with 60 cells per replicate). ** p<0.01, ns p>0.05. P: Physiological T3; 0: 0 nM T3; 100: 100 nM T3.

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques: Over Expression, Expressing, Transfection, Fluorescence, Microscopy, Mutagenesis

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: MED1 overexpression increases the percentage of A263V-expressing cells with aggregates, while MED1 knockout and T3 supplementation decrease the percentage of cells with aggregates. mCherry-tagged WT, A263V, and A263S TRα1 expression plasmids were either single-transfected or cotransfected with GFP-MED1 or GFP-MED13 into HeLa cells, MED1+/+ MEFs, and MED−/− MEFs, as indicated. Cells were analyzed by fluorescence microscopy 24 hours post-transfection. (A) Representative images are shown for different transfection schemes. Scale bar = 10 μm. (B) Bars represent the average percent of cells with TR aggregates for each transfection scheme under physiological levels of T3. (C) Bars represent the average percent of cells with TR aggregates for each transfection scheme under depleted or elevated levels of T3. The presence or absence of aggregates was assessed qualitatively by scoring cells as having “no aggregation” (homogeneous distribution of TR without any bright fluorescent foci) or having “aggregation” (bright fluorescent foci of TR, ranging from speckles to larger aggregates). (D) Bars represent the average percent of cells with TR aggregates for MED1+/+ or MED1−/− MEFs under physiological levels of T3. Error bars indicate +/− SEM (n=3 biologically independent replicates, with 60 cells per replicate). *p<0.05, **p<0.01, ns p>0.05). (E) HeLa cells cotransfected with the aggresome marker GFP-250 and either wild-type or A263V TRα1 were analyzed for colocalization of aggregates and aggresomes by confocal microscopy.

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques: Over Expression, Expressing, Knock-Out, Transfection, Fluorescence, Microscopy, Marker, Confocal Microscopy

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: Overexpression of MED1 rescues the decreased intranuclear mobility of A263V TRα1. mCherry tagged WT, A263V, P398R, and C392X TRα1 expression plasmids were either single-transfected or cotransfected with GFP-MED1 into HeLa cells. Strip-FRAP was performed 24 h post transfection on 20 nuclei with a stimulation bleaching line near the middle of each nucleus. (A) Representative nuclei prior to bleach (pre-bleach), directly after bleaching terminated (bleach), 1 s post-bleach (+1s), and at the end of the recovery (final). Graphs represent the average normalized fluorescence intensity for each time point for A263V (B), P398R (C), and C392X TRα1 (D). Error bars indicate +/− SEM (n=3 biologically independent replicates, with 20 nuclei per replicate). * p<0.05.

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques: Over Expression, Expressing, Transfection, Stripping Membranes, Fluorescence

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: Intranuclear FRAP profile for wild-type (WT) and A263V TRα1 in HeLa cells under physiological T3 levels. a

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques:

Journal: Molecular and cellular endocrinology

Article Title: Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome

doi: 10.1016/j.mce.2022.111781

Figure Lengend Snippet: TR-mediated T3-dependent reporter gene transactivation is diminished in RTH mutants. (A) HeLa cells were cotransfected with expression plasmids for wild-type (WT) GFP-TRα1 and the RTH mutants, as indicated, TRE (DR+4)-firefly luciferase reporter, and Renilla luciferase internal control, in the presence or absence of 100 nM T3. Data are presented as relative firefly/Renilla luciferase activity (Relative Luciferase) normalized to -T3. Error bars indicate ± SEM (n=3 biologically independent replicates of 8 wells per treatment). ***p<0.001, **p<0.01, ns p>0.05.

Article Snippet: Cell culture and transient transfection HeLa cells (#CCL-2; American Type Culture Collection) were grown in minimum essential medium (MEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C under 5% CO 2 and 98% humidity.

Techniques: Expressing, Luciferase, Control, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

doi: 10.1091/mbc.e20-12-0797

Figure Lengend Snippet: FIGURE 3: zfCx43∆256-289 forms larger and more GJ plaques and exhibits longer protein half-lives and increased dye transfer in cultured cells. (A) Representative IF images of Cx43 (magenta) and DAPI (blue) showing GJ plaques in the membranes of HeLa cells 24 h posttransfection. (B) Plaque size analyses plotted as total fluorescence intensity indicate that Cx43∆256-289-expressing cells on average exhibit approximately two times the area (plaque number and size combined) of GJs in their plasma membranes compared with WT Cx43- expressing cells (n = 258 WT and 190 Cx43∆256-289 plaques, p ≤ 0.05). (C) Representative Western blot of Cx43 protein half-life at 0 and 6 h after cycloheximide treatment. (D) Quantification indicates that Cx43∆256-289 protein remains stable even after 6 h, while WT Cx43 protein is degraded with a typical half-life of approximately 4 h (n = 3 transfected cultures, p ≤ 0.05). (E) Representative images of LY scrape-loading dye transfer assays of untransfected MDCK cells (–), MDCK cells transfected with WT Cx43, and MDCK cells transfected with Cx43∆256-289. (F) Quantification of distances LY diffused away from the scrapes indicates that cells expressing Cx43∆256-289 exhibit significantly increased dye transfer compared with cells expressing WT Cx43 or cells without Cx43 protein (n = 3 transfected cultures, p ≤ 0.05; error: SD).

Article Snippet: Cell culture and transient transfections HeLa (GJ deficient; Cat. No. CCL2; American Type Culture Collection [ATCC]) (Elfgang et al., 1995) and MDCK cells (GJ deficient; Cat. No. NBL-2; ATCC) (Dukes et al.., 2011) were maintained in lowglucose DMEM (Cat. No. SH30021.01; Hyclone) supplemented with 50 I.U./ml penicillin and 50 μg/ml streptomycin (Cat. No. 30-001- C1; Corning), 2 mM ʟ-glutamine (Cat. No. 25-005-C1; Mediatech), and 10% fetal bovine serum (FBS) (Cat. No. S11150; Atlanta Biologicals) at 37°C, 5% CO2, and 100% humidity.

Techniques: Cell Culture, Fluorescence, Expressing, Clinical Proteomics, Western Blot, Transfection