Journal: STAR Protocols

Article Title: An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

doi: 10.1016/j.xpro.2022.101288

Figure Lengend Snippet:

Article Snippet: HEK293T , ATCC , ACS-4500.

Techniques: Recombinant, Plasmid Preparation, Gel Extraction, Purification, Sequencing, Amplification, Software, Membrane, Pore Size, Spectrophotometry, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Antibody validation for Western blot: By the user, for the user

doi: 10.1074/jbc.RA119.010472

Figure Lengend Snippet: Validation of IDH1 antibody using purified recombinant protein in multicolor and chemiluminescent Western blotting. Multicolor and chemiluminescent Western blottings were performed using 10% Bis-Tris SDS-polyacrylamide gel and MOPS buffer system to validate the IDH1 antibody using a purified recombinant IDH1 protein (0.16 μg) containing a c-Myc tag in addition to HEK293T and HeLa whole-cell lysates. A, c-Myc protein tag present on the purified IDH1 recombinant protein is detected in the 700-nm channel ( red ) at 50 kDa via mouse anti-c-Myc antibody (ab32;1 μg/ml) using IRDye 680RD goat anti-mouse IgG (H + L) for detection. Some overspill of the recombinant protein into neighboring lanes is observed ( white box ). B, IDH1 recombinant protein and endogenous IDH1 protein, present in HEK293T and HeLa, is detected in the 800-nm channel ( green ) at 55 and 50 kDa, respectively, using rabbit anti-IDH1 antibody (ab172964; 1.2 μg/ml) and IRDye 800CW goat anti-mouse IgG (H + L) for detection. C, when both 700- and 800-nm channels are displayed, the signal from ab32 and ab172964 overlaps at 50 kDa, identifying the c-Myc–tagged IDH1 protein. No overlap is seen for the endogenous IDH1 present in HEK293T and HeLa whole-cell lysates. A–C , lysates loaded per lane are as follows: 20 μg of blocking buffer: Odyssey blocking buffer (TBS); imager: Odyssey® CLx; resolution: 169 μm; intensity: auto mode. Chameleon TM Duo pre-stained protein ladder for accurate sizing of protein bands. D, single blot was split into two halves ( green line ) to be incubated with either rabbit anti-IDH1 antibody (ab172964; 0.115 μg/ml) or the corresponding rabbit monoclonal IgG isotype control (ab172730; 0.166 μg/ml) to detect the endogenous IDH1 protein present in HeLa and HEK293T as well IDH1 recombinant protein. Both halves were incubated with HRP-conjugated goat anti-mouse IgG (H + L). E, single blot was split into two halves ( green line ) to be incubated with either mouse anti-c-Myc antibody (ab32; 1 μg/ml) or the corresponding mouse monoclonal IgG1 isotype control (ab18443; 1 μg/ml) to detect c-Myc protein tag present on the purified IDH1 recombinant protein but absent in HEK293T and HeLa whole-cell lysates. Both halves were incubated with HRP-conjugated goat anti-rabbit IgG (H + L). Blots were detected with WesternSure® PREMIUM chemiluminescent substrate (LI-COR 926–95000) and imaged on an Odyssey® Fc with the following resolution: 125 μm and exposure of 2 min. Lysate loaded per lane: 20 μg; protein ladder: WesternSure® pre-stained chemiluminescent protein ladder (LI-COR 926-980000); blocking buffer: intercept blocking buffer (TBS); intercept T20 (TBS) antibody diluent.

Article Snippet: Isocitrate dehydrogenase (IDH1) (NM_005896) human recombinant protein (OriGene no. TP310582), IDH1 (NM_005896) human overexpression lysate supplied with parental HEK293T lysate (OriGene no. LY401782; HEK293T LY500001; lot no. 0076CF), and IDH1 knockout cell lysate (supplied with parental HeLa control lysate) (Origene no. LC810112, LC810Hela; lot no. 1601) were mixed with either 2× protein loading buffer (PLB) (LICOR no. 928-40004) or 2× SDS buffer (OriGene) and denatured by boiling at 97 °C for 5 min.

Techniques: Biomarker Discovery, Purification, Recombinant, Western Blot, Blocking Assay, Staining, Incubation, Control

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 2. In vitro knockdown of human RHO-T17M expression. (A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and SaCas9/17-Sg2 were appeared to cut the mutant sequence specifically, the full-length amplicon was 760 bp, the two truncated amplicons were 510 bp and 250 bp, respectively. (C) The cutting efficacy of two sgRNAs with SaCas9 determined by TA and Sanger sequencing in 293T cells. (D) Rhodopsin expression reduction was determined by WB in RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, comparing to the RHOwt cells with 17-Sg1 and -Sg2 plasmid. (E) Densitometric analysis of immunoblots performed on RHOwt and RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, respectively. The experiment was performed in triplicate and presented as mean ± SEM, the significance was calculated using two-tailed paired t-test, ns = not significant, *p<0.05.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: In Vitro, Knockdown, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 6. Expression of the mutant human RHO allele after gene editing with SaCas9/17-Sg2 in vitro. (A) Schematic view of the different human RHO gene variants created by gene editing. (Top) Map of the pEGFPN1 vector used to overexpress these variants. (Bottom) The description of variants at DNA and protein level. (B) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-WT, RHO-T17M, and four edited RHO-T17M variants, 1 week after transfection. Scale bar = 10 μm. (C) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-5m and four edited RHO-5m variants, 1 week after transfection. Scale bar = 10 μm. (D–F) The number of GFP+ cells and percentage of GFP+ cells expressing rhodopsin per random sight. Nuclei were stained blue by DAPI. Scale bar = 200 μm.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Mutagenesis, In Vitro, Plasmid Preparation, Transfection, Staining

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 10. Examination of SaCas9/17-Sg2 off-target effects in human gDNA using WGS. Identification of SNVs (A) and indels (C) in 293T cells transfected with 17-Sg2 plasmid at the WGS level. The type of SNVs (B) and indels (D) in 293T cells transfected with 17-Sg2 plasmid and untreated cells at the WGS level.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation

Journal: Human molecular genetics

Article Title: The utrophin A 5'-UTR drives cap-independent translation exclusively in skeletal muscles of transgenic mice and interacts with eEF1A2.

doi: 10.1093/hmg/ddp591

Figure Lengend Snippet: Figure 3. eEF1A-2 interacts with the utrophin A 50-UTR. (A) Northwestern analysis of control and cardiotoxin (CTX)-injected tibialis anterior (TA) muscles using a [a-32P]UTP-labeled RNA probe corresponding to nucleotides 147–363 of the utrophin A 50-UTR. Note the presence of several bands in regenerating muscles. Blot is representative of experiments performed with muscles of three mice. (B) RNA-affinity chromatography isolation of utrophin A 50-UTR-binding proteins. Precleared extracts from cardiotoxin-treated TA muscles were incubated with agarose beads coated with biotinylated utrophin A 50-UTR RNA (147–363) or agarose beads alone. Beads were washed exten- sively, eluted by boiling and resolved by SDS–PAGE. Sypro Ruby stained gel shows a 50 kDa protein species that was identified as eukaryotic elongation factor 1A2 (eEF1A2) by mass spectrometry analysis. (C) Samples prepared as in (B) were separated by SDS–PAGE, transferred to PVDF membrane, and western blot was performed using an anti-EF1A antibody. This antibody detects both eEF1A1 and eEF1A2 isoforms. eEF1A was detected in CTX muscle lysate incubated with the utrophin A 50-UTR biotinylated probe, but not to a no RNA control or an unrelated biotinylated RNA probe (correspond- ing to the utrophin A 30-UTR). (D) Biotinylated 50-UTR probe (147–363) does not bind to eEF1A from HEK293T protein lysate (HEK).

Article Snippet: To confirm eEF1A2 as an interacting protein, we performed RNA-affinity chromatography using 15 mg of biotinylated RNA and 1.6 mg of TA muscle or HEK293T cell extracts, transferred proteins from the gel to a PVDF membrane, and performed western blot using an eEF1A antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Injection, Muscles, Labeling, Chromatography, Isolation, Binding Assay, Incubation, SDS Page, Staining, Mass Spectrometry, Membrane, Western Blot