Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: Truncating variants of NFKB1 impair autophagy (A) MitoSOX staining of untreated NFKB1 R157∗/R157∗ and WT THP-1 monocytes was analyzed by flow cytometry. Quantifications and representative plots are shown. (B) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS, and MitoTempo was applied 1 h before ATP. Secreted mature IL-1β was detected by ELISA. (C) ρ 0 or conventional NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS and secreted mature IL-1β was detected by ELISA. (D) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS for indicated times and SQSTM1 was blotted from lysates. Quantifications, representative blots, and TPL are shown. (E) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were inhibited with E-64d and pepstatin A (abbreviated as EP) 1 h prior to LPS (for indicated times). LC3 was blotted from cell lysates. Quantifications, representative blot, and TPL are shown. (F and G) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were treated with LPS and (F) 3-MA (6 h 45 min) or (G) rapamycin (17 h 45 min) and ATP (for the last 45 min). Secreted mature IL-1β was detected by ELISA and is expressed as fold change relative to the respective cells stimulated with LPS and ATP. (H) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS (18 h). Cells staining positive for Hoechst (nuclear stain) and autophagic vesicle marker were analyzed by flow cytometry. Quantitation and representative plots are shown. (I) Schematic representation of the effects of truncating NFKB1 mutations on macroautophagy and NLRP3 inflammasome activation. Wilcoxon test (A), one-way RM-ANOVA (B), or two-way ANOVA (C–H) followed by Dunnett’s (B), Šidak’s (D–G), or Tukey’s test (C and H). The data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) n = 8; (B) HMDMs: 1 control, 3 variant carriers, THP-1 n = 4; (C–E) THP-1 n = 4; (D) HMDMs: 2 controls, 4 variant carriers; (F–H) THP-1 n = 4. See also Figure S4 .

Article Snippet: For staining with LC3 antibody (Novus Biologicals NB100-2331) THP-1 monocytes were centrifuged and lyzed in sucrose lysis buffer (250 mM Sucrose, 1 mM EDTA, and 1x protease inhibitor).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Quantitation Assay, Activation Assay, Control, Variant Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet:

Article Snippet: For staining with LC3 antibody (Novus Biologicals NB100-2331) THP-1 monocytes were centrifuged and lyzed in sucrose lysis buffer (250 mM Sucrose, 1 mM EDTA, and 1x protease inhibitor).

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Cell Culture, Molecular Weight, Staining, Sequencing, Modification, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Transfection, Quantitation Assay, Mutagenesis, Software, CRISPR, Variant Assay, Cytometry

Journal: Amyotrophic lateral sclerosis & frontotemporal degeneration

Article Title: Combination of ciprofloxacin/celecoxib as a novel therapeutic strategy for ALS.

doi: 10.1080/21678421.2022.2119868

Figure Lengend Snippet: Figure 3. ALS-pathology related NDE biomarker levels in ALS vs. control samples. Wilcox test comparing neural-derived blood exosomal biomarkers: TDP-43 (A) and LC3 (B) between patients with ALS and healthy volunteers (N ¼ 35 for ALS and 25 for control per group) as obtained in a baseline study.

Article Snippet: LC3 and TDP-43 were quantitatively measured in the NDE protein extracts using Human LC3 DuoSet ELISA (R&D Systems, Cat. No DY8558-05), and ELISA kit for TDP43 (MyBioSource, Cat No MBS2019049) per manufacturer’s instructions.

Techniques: Biomarker Discovery, Control, Derivative Assay

Journal: Amyotrophic lateral sclerosis & frontotemporal degeneration

Article Title: Combination of ciprofloxacin/celecoxib as a novel therapeutic strategy for ALS.

doi: 10.1080/21678421.2022.2119868

Figure Lengend Snippet: Figure 4. ALS-pathology related biomarker levels in PrimeC treated ALS patients over time. (A,B) Average ALS-pathology related serum NDE marker levels over time (pre in red, interim in light blue, and end of study in dark blue). (A) TDP-43 levels. (B) LC3 levels. (C,D) Average serum NFL (C) and pNFH (D) over time (pre in red, interim in light blue, and end of study in dark blue). P values tests whether the means at pre-treat are similar compared to the mean at end-of-treat, based on a mixed model for repeated measures.

Article Snippet: LC3 and TDP-43 were quantitatively measured in the NDE protein extracts using Human LC3 DuoSet ELISA (R&D Systems, Cat. No DY8558-05), and ELISA kit for TDP43 (MyBioSource, Cat No MBS2019049) per manufacturer’s instructions.

Techniques: Biomarker Discovery, Marker

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 2. Localization of Ulk1 and LC3 in rat kidneys. Normal rat kidney sections were stained with Alexa 488-phalloidin, an antibody against UNC-51-like kinase 1 (Ulk1) or microtubule-associated protein 1 light chain 3 (LC3). Red signals for Ulk1 (E), (H) or LC3 (K) were merged with green signals for phalloidin (D), (G) and (J) in the same section (F), (I) and (L). Magnification, 200 (A)–(C) and 400 (D)–(L); scale bars, 100 mm (A)–(C) and 50 mm (D)–(L). OSOM, outer stripe of outer medulla; ISOM, inner stripe of outer medulla. (*), glomeruli.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Staining

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 3. Pharmacological effects of everolimus in normal kidney epithelial cell lines.(A) Human kidney 2 (HK-2), Madin–Darby canine kidney (MDCK), and normal rat kidney epithelial (NRK-52E) cells were treated with everolimus (Eþ, 100 nM) or vehicle (E) for 48 h. After treatment, whole-cell extracts were obtained and examined with Western blotting using antibodies against LC3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH was used as a loading control to determine the relative densities of the bands in each lane. The ratio of the density of everolimus-treated cells to that of vehicle-treated cells was calculated. Each column represents the mean7S.E.M. (n¼3; *Po0.05, significantly different). (B) After the cells were exposed to everolimus for 48 h, viable cell number was determined. The viable cell number of the cells treated with vehicle alone was regarded as 1.0. Each column represents the mean7S.E.M. of 3 wells. *Po0.05, **Po0.01, significantly different. (C) Protein expression of Ulk1 in NRK-52E cells was examined with Western blotting using an antibody against Ulk1. Cells were transfected with small interfering RNA (siRNA) targeting rat Ulk1 (siUlk1) or negative-control siRNA (siControl). (D)–(G) After transfection with siControl or siUlk1, cells were treated with vehicle alone or everolimus at various concentrations. The viability of the control cells at 0 h was regarded as 1.0. Each point represents the mean7S.E.M. (n¼3).

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Western Blot, Control, Expressing, Transfection, Small Interfering RNA, Negative Control

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 4. Examination of LC3-II levels in rat skeletal muscles after starvation. (A) Whole-tissue homogenates were prepared from skeletal muscles after a 0- or 48-h starvation and subjected to western blotting using antibodies against LC3 and GAPDH. (B) The relative densities of the bands in each lane were determined, and the ratio of the density of the cells of starved rats to that of the cells of control rats was calculated. GAPDH was used as an internal standard. **Po0.01, significantly different from control rats.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Muscles, Western Blot, Control

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 5. Effects of ischemia-reperfusion on autophagy in rat kidneys. Rats were subjected to ischemia-reperfusion. Kidneys were collected 1, 2, 4, and 7 days after surgery. Rats in the control group were sham-operated, and kidneys were collected 2 days after surgery. The number of rats in each group was 6. (A)–(E) Whole-kidney homogenates were subjected to Western blotting. The expression levels of LC3, total Ulk1, phosphorylated Ulk1 (p-Ulk1), total ribosomal protein S6, phosphorylated S6 (p-S6), and GAPDH were examined. (B)–(E) The relative densities of the bands in each lane were determined. (F), (G) Renal function was assessed by measuring plasma creatinine concentration (PCr) and blood urea nitrogen levels. Multiple comparisons were performed using Dunnett’s 2-tailed test. *Po0.05, **Po0.01, significantly different from control rat kidneys.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Control, Western Blot, Expressing, Clinical Proteomics, Concentration Assay

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 6. Effects of everolimus in rat kidneys after treatment with ischemia-reperfusion. Rats were administered everolimus (Eþ, 2 mg/kg per day) or vehicle (E) for 7 days. On day 5 of administration, rats were also subjected to ischemia-reperfusion. Kidneys were collected on day 2 post-treatment. In a control group, rats were sham-operated, and kidneys were collected on day 2 after surgery. The expression levels of LC3, total Ulk1, p-Ulk1, total S6, p-S6, and GAPDH were examined using Western blot analysis (A). The relative densities of the bands in each lane were determined (B)–(D). *Po0.05, **Po0.01, yPo0.05, yyPo0.01 show significant difference.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Control, Expressing, Western Blot

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 7. Effects of everolimus on renal functions after ischemia-reperfusion. Levels of PCr) (A), kidney weight (B), LC3 (C)–(E), kidney injury molecule-1 (Kim-1; (F)–(H)), single-stranded DNA (ssDNA) (I)–(K), and Ki-67 (L)–(N) were examined after concomitant treatment with everolimus and ischemia-reperfusion. Green signals for phalloidin are merged with red signals for LC3 (C), (D); Kim-1 (F), (G); ssDNA (I), (J); or Ki-67 (L), (M). Magnification, 400 ; scale bars, 50 mm; (*), glomeruli. The numbers of dot signals (for LC3, (E)); stained proximal tubules (for Kim-1, (H)); or stained nuclei (for ssDNA, (K); Ki-67, (N)) were counted. Each column represents the mean7S.E.M. (*Po0.05, **Po0.01, yPo0.05, and yyPo0.01 show significant differences).

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Staining

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 8. Effects of cisplatin on autophagy in rat kidneys. Rats were intraperitoneally administered cisplatin (5 mg/kg body weight). Kidneys were collected 1, 2, 4, and 7 days after administration. As a control group, rats were administered vehicle, and kidneys were collected 2 days after administration. The number of rats in each group was 6. (A)–(E) Whole-kidney homogenates were subjected to Western blotting. The expression levels of LC3, total Ulk1, p-Ulk1, total S6, p-S6, and GAPDH were examined. (B)–(E) The relative densities of the bands in each lane were determined. (F), (G) Renal function was assessed by measuring PCr and blood urea nitrogen levels. Multiple comparisons were performed using Dunnett’s 2-tailed test. *Po0.05, **Po0.01, significantly different from control rat kidneys.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Control, Western Blot, Expressing

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 9. Effects of everolimus in rat kidneys after treatment with cisplatin. Rats were administered everolimus (Eþ, 2 mg/kg per day) or vehicle (E) for 7 days. On day 5 of administration, rats were also administered cisplatin. Kidneys were collected on day 2 post-treatment. As a control group, rats were administered the vehicle, and kidneys were collected on day 2 after administration. The expression levels of LC3, total Ulk1, p-Ulk1, total S6, p-S6, and GAPDH were examined using Western blot analysis (A). The relative densities of the bands in each lane were determined (B)–(D). *Po0.05, yPo0.05 show significant difference.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Control, Expressing, Western Blot

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 10. Effects of everolimus on renal functions after cisplatin treatment. Levels of PCr (A), kidney weight (B), LC3 (C)–(E), Kim-1 (F)–(H), single-stranded DNA (ssDNA) (I)– (K), and Ki-67 (L)–(N) were examined after concomitant treatment with everolimus and cisplatin. Green signals for phalloidin are merged with red signals for LC3 (C), (D), Kim-1 (F), (G), ssDNA (I), (J), or Ki-67 (L), (M). Magnification, 400 ; scale bars, 50 mm; (*), glomeruli. The numbers of dot signals (for LC3, (E)), stained proximal tubules (for Kim-1, (H)), or stained nuclei (for ssDNA, (K); Ki-67, (N)) were counted. Each column represents the mean7S.E.M. (*Po0.05 and **Po0.01 show significant differences).

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Staining

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 11. Urinary LC3 concentration after treatment with everolimus. (A) Western blotting of LC3 protein in rat urine using urine collected from rats after co-treatment with everolimus and cisplatin. Proteins were probed with 2 anti-LC3 antibodies (Detection, the detection antibody in ELISA; Capture, the capture antibody in ELISA). Two bands (arrowheads) were observed with both antibodies. (B) A typical standard curve using recombinant LC2 protein was made using serial dilutions from a 500 ng/mL concentration. R2¼0.98 represents a high correlation coefficient. Each point represents the absorbance at 450 nm minus the value of assay blank and is expressed as the mean7S.E.M. of 3 wells. (C), (D) Rats were administered everolimus (2 mg/kg) or vehicle subcutaneously for 7 days. On the fifth day, the rats were subjected to ischemia- reperfusion (C) or treated with cisplatin (D). Urinary LC3 concentration (nanograms per milligram creatinine) in each rat was determined (n¼6, each). *Po0.05, significantly different from vehicle treated rats.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: European journal of pharmacology

Article Title: Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury.

doi: 10.1016/j.ejphar.2012.09.010

Figure Lengend Snippet: Fig. 12. The hypothesized mechanisms of mammalian target of rapamycin (mTOR) inhibitor action in proximal tubular epithelial cells. (A) During the recovery phase of nephrotoxicants-induced tubular injury, the mTOR pathway in proximal tubular cells is activated to promote cell proliferation. (B) Everolimus treatment exerts antiproliferative effects by blocking cell survival pathways. The quantification of urinary LC3 protein could be beneficial in predicting outcomes of treatment with mTOR inhibitors.

Article Snippet: Two antibodies specific for LC3 (Novus Biologicals NB100-2331B and Cell Signaling Technology #2775) were used as primary antibodies.

Techniques: Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: (+)-Lipoic Acid Reduces Lipotoxicity and Regulates Mitochondrial Homeostasis and Energy Balance in an In Vitro Model of Liver Steatosis

doi: 10.3390/ijms241914491

Figure Lengend Snippet: Computerized analysis of LC3A and % of autophagic cells. * vs. CTRL (*** p < 0.001, **** p < 0.0001); § vs. PA:OA ( §§§ p < 0.001, §§§§ p < 0.0001). ( A ) quantification of % of autophagic cells; ( B ) Representative images of autogaphy-positive cells. The figures presented are representative of at least three independent experiments. Scale bars in ( B ) 50 μm.

Article Snippet: Subsequently, cells were incubated with human LC3A antibody FITC-conjugated (NB100-2331F, Novus Biologicals) at a dilution of 1:200 overnight at 4 °C.

Techniques:

Journal: Frontiers in cell and developmental biology

Article Title: ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

doi: 10.3389/fcell.2021.716208

Figure Lengend Snippet: FIGURE 1 | Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) ORF3a triggers incomplete autophagy. (A–B) SARS-CoV-2 infection triggers incomplete autophagy. HeLa-hACE2 (A) or Vero-E6 (B) cells were infected with SARS-CoV-2 (MOI = 1) and cell lysates were collected at indicated time point post-infection for immunoblotting (IB) with indicated antibodies. (C) Screening of SARS-CoV-2-encoded proteins for LC3-I to LC3-II conversion in HeLa cells. (D,E) SARS-CoV-2 ORF3a induces incomplete autophagy. HeLa-vector and HeLa-ORF3a stable cells were treated with or without BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). HeLa cells were transfected with increasing amount ORF3a expressing plasmid and the cell lysates were collected for IB with indicated antibodies at 48 post-transfection (E). (F) ORF3a expression modulates autophagy in Vero-E6 cells. (G) ORF3a blocks SQSTM1/p62 degradation upon starvation. HeLa-vector or HeLa-ORF3a stable cells were treated by serum starvation for 4 h and the cell lysates were collected for IB with indicated antibodies. (H,I) SARS-CoV-2 ORF3a induces LC3 puncta formation. HeLa-vector or HeLa-ORF3a cell line were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (H) and quantified (I). Scale bar, 15 µm. Arrow: representative autophagosomes. (J,K) HeLa-mRFP-GFP-LC3 stable cells were transfected with ORF3a or vector control and transfected cells were fixed and the LC3 puncta were captured (J,K) quantified as indicated. Yellow puncta, autophagosomes; Red puncta, autolysosomes; Arrow, representative autophagosomes. p62/GAPDH or LC3-II/GAPDH levels were quantified by the band intensity in (A–G). Similar results were obtained by three independent experiments. Mean ± SEM; n = 50; ***p < 0.001 and ****p < 0.0001 by Student’s t test in panels (I,K).

Article Snippet: For the LC3 conversion assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749).

Techniques: Infection, Western Blot, Plasmid Preparation, Transfection, Expressing, Control

Journal: Frontiers in cell and developmental biology

Article Title: ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

doi: 10.3389/fcell.2021.716208

Figure Lengend Snippet: FIGURE 4 | SARS-CoV ORF3a cannot induce autophagy. (A) Sequence alignment between SARS-CoV-2 ORF3a and SARS-CoV ORF3a. (B–D) SARS-CoV ORF3a does not affect autophagy. HeLa-vector, HeLa-ORF3aSARS-CoV-2, or HeLa-ORF3aSARS-CoV cell lines were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (B) and quantified (C). Scale bar, 15 µm. Arrow: representative autophagosomes. Mean ± SEM; n = 50; ns and ****p < 0.0001 by one-way ANOVA and Bonferroni’s post hoc test. HeLa-vector or HeLa-ORF3aSARS-CoV cell lines were treated with BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). (E) ORF3aSARS-CoV-2 but not ORF3aSARS-CoV interacts with endogenous UVRAG. HEK293T cells were transfected with Flag-ORF3aSARS-CoV or Flag-ORF3aSARS-CoV-2 and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48h post-transfection. (F–H) SARS-CoV ORF3a does not affect the formation of UVRAG complex (F), Beclin 1 complex (G), or Atg14 complex (H). HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48 h post-transfection.

Article Snippet: For the LC3 conversion assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749).

Techniques: Sequencing, Plasmid Preparation, Transfection