Journal: Frontiers in Immunology

Article Title: Ferritin Light Chain: A Candidate Autoantigen in Immuno-Related Pancytopenia

doi: 10.3389/fimmu.2022.851096

Figure Lengend Snippet: Analysis of FTL levels in patients with IRP, and the correlation between the expression level of FTL and clinical indicators. (A) Flow cytometry plots of FTL. (B) Results of the FCM assay of FTL on surface of BMMNCs. (C) Results of the FCM assay of FTL on surface of CD34 + BM cells. (D) Results of the FCM assay of FTL on surface of CD235a + BM cells. (E) Results of the FCM assay of FTL on surface of CD15 + BM cells. (F) Q-PCR analysis of FTL-mRNA. (G) FTL expression level was analyzed by ELISA. (H) Western Blot analysis of FTL protein levels. (I) The correlation between the expression level of FTL and clinical indicators.

Article Snippet: We purchased a human FTL ELISA kit (Sino Biological Inc., China) to detect FTL levels in plasma samples.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Comparative proteomics reveals a diagnostic signature for pulmonary head‐and‐neck cancer metastasis

doi: 10.15252/emmm.201708428

Figure Lengend Snippet: A Volcano plot relating adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates. Red (higher expression in HNSCC) and blue (higher expression in SQCLC) dots indicate significantly regulated proteins ( P < 0.05). B Two‐class comparison of genetic dependencies from a publically available genome‐scale CRISPR‐Cas9 screen of HNSCC (12 cell line) versus SQCLC (10 cell lines) identified a subset of differentially expressed proteins that were also differential dependencies in these tumour types. The x ‐axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines. Positive effect size indicates a greater mean dependency in HNSCC; negative effect size indicates a greater mean dependency in SQCLC. The y ‐axis represents the statistical significance of differential enrichment calculated as ‐log10( P ‐value) from a two‐sided t ‐test. The P ‐values used for this plot are uncorrected for multiple hypothesis testing. Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study. C, D Immunohistochemical analysis of the expression of HMGCS‐1 ( P = 0.0014), FTL ( P = 0.0001) (C), LGALS7 ( P = 0.0001) and FAM83H ( P = 0.0001) (D) in an independent cohort of 212 SQCLC and 343 HNSCC cases. Scale bar indicates 100 μm. Shown are mean values and standard deviation. Statistical significance was assessed using Wilcoxon–Mann–Whitney test. E Pathway enrichment analysis for proteins differentially expressed in HNSCC and SQCLC.

Article Snippet: Briefly, 2‐μm tissue sections were incubated in EnVision Flex Target Retrieval Solution, pH high or low (Dako) followed by incubation of primary antibodies against CK5/6 (Dako, prediluted, high), CK7 (Dako, prediluted, high), p63 (Bio‐Genex, prediluted, high), TTF‐1 (Dako, prediluted, high), CD34 (Dako, prediluted, low), CK19 (Dako, prediluted, high), FTL (Atlas antibodies, 1:1,000, high), FAM83H (1:500, low), LGALS7 (Abcam, 1:20,000, high) or HMGCS1 (Atlas antibodies, 1:200, low) at room temperature for 20 min. Polymeric secondary antibodies coupled to HRPO peroxidase (EnVision Flex + , Dako) and DAB (Dako) were applied to visualise the sites of immunoprecipitations.

Techniques: Expressing, Multiplex sample analysis, Comparison, CRISPR, Immunohistochemical staining, Standard Deviation, MANN-WHITNEY

Journal: Bioconjugate chemistry

Article Title: Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging.

doi: 10.1021/acs.bioconjchem.8b00004

Figure Lengend Snippet: Figure 1. Illustration of FTL-X variant construction and production in E. coli. Human FTL was modified by PCR cloning method to incorporate amber stop codon (TAG) at desired position in the FTL cDNA. The FTL-X plasmid was cotransformed along with the pEVOL-AzF vector, which encodes the tRNA/aaRS pair for para-azido-L-phenylalanine (pAzF),33 into competent E. coli. Large scale production was performed by IPTG induction in the presence of pAzF. FTL-X was extracted by sonication and heat precipitation followed by purification by FPLC.

Article Snippet: A full-length cDNA for hFTL (SC120074) was purchased from Origene (Rockville, MD) and cloned into the bacterial expression vector, pRSET A (Thermo Fisher Scientific, Philadelphia, PA).

Techniques: Variant Assay, PCR Cloning, Plasmid Preparation, Sonication

Journal: Bioconjugate chemistry

Article Title: Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging.

doi: 10.1021/acs.bioconjchem.8b00004

Figure Lengend Snippet: Figure 2. Characterization of FTL-5X and FTL-5X/anti-ICAM conjugates. (a) Western blot (staining for hFTL), (b) fluorescence scan, and (c) SDS-PAGE analysis of FTL-5X conjugated to AlexaFluor488 labeled anti-ICAM at different molar ratios. Lane numbers indicate the following: (1) FTL-5X, (2) anti-ICAM, (3) FTL-5X/anti-ICAM at molar ratio of 1 to 2, and (4) FTL-5X/anti-ICAM at molar ratio of 1 to 10. TEM images and size analysis of FTL-5X and antibody conjugates. TEM images of (e) FTL-5X and (f) FTL-5X/anti-ICAM. Scale bar: 100 nm. Particle size analysis (size distribution by number) obtained by DLS for (d) FTL-5X, and FTL-5X/anti-ICAM, as well as their (g) mean size and polydispersity index (PDI).

Article Snippet: A full-length cDNA for hFTL (SC120074) was purchased from Origene (Rockville, MD) and cloned into the bacterial expression vector, pRSET A (Thermo Fisher Scientific, Philadelphia, PA).

Techniques: Western Blot, Staining, SDS Page, Labeling, Particle Size Analysis

Journal: Nature communications

Article Title: Cryo-electron tomography pipeline for plasma membranes.

doi: 10.1038/s41467-025-56045-z

Figure Lengend Snippet: Fig. 4 | Subtomogram averaging and contextual analysis show sub-nanometer detail is preservedin isolated plasma membranes. a Projection of 21z-slices from a tomogram of an isolated plasma membrane representative of the 111 tomograms used in this analysis. 80S ribosomes (black arrows) are frequently found in unroofed HEK293 cells overexpressing dynamin-1(K44A). b Rotated views (top) and a clipped view (bottom) of a consensus subtomogram average filtered according to the local resolution. c Fourier shell correlation (FSC) profiles obtained from sub- tomogram averages. The nominal resolution is reported at FSC = 0.143. d, e Classification of the set of well-aligning particles obtained subtomogram averages of the 80S ribosome in non-rotated and rotated states. In (d), tRNA occupying the P, P/E, and A/P sites are indicated in orange, pink, and blue,

Article Snippet: For FerriTag experiments, HEK293 cellswere transfectedwith FTL (Ferritin light chain, Addgeneplasmid# 100750), FRB-mCherry-FTH1 (Ferritin Heavy Chain, Addgene Plasmid #100749), and one of either HIP1R-GFP-FKBP (Addgene Plasmid #100752) or GFP-FKBP-LCa (Addgene Plasmid #59353)16 using the Lonza Nucleofector and kit V (Lonza #VCA-1003) immediately prior to coverslip seeding (western blot, Fig. S11a, b).

Techniques: Isolation, Clinical Proteomics, Membrane