anti flnb  (Proteintech)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Screening and identification of protein 29 of Echinococcus granulosus interacting molecules

doi: 10.3389/fcimb.2025.1560436

Figure Lengend Snippet: Changes in mRNA and protein expression levels of seven molecules that interact with Eg.P29 in different groups. (A) mRNA expression levels of ARPC1A, LIMA1, FLNB, MYH10, ACTN4, ACTG1 , and VCL . HEK-293T cells were transfected with Eg.P29 /pEGFP-C1vector and the corresponding empty vector for 24 hours. The total RNA was extracted and the mRNA level of specific molecules was shown by PR-qPCR. GAPDH was used as the reference gene, and the 2 −δδCt method was used to evaluate gene expression. All RT-qPCR experiments were conducted independently six times. Error bars represent the standard error of the mean (SEM). ns means no statistical difference, * P <0.05, ** P <0.01, *** P <0.001, and **** P <0.0001. (B) The protein expression levels of ACTN4, LIMA1, ARPC1A, FLNB, ACTG1, VCL, and MYH10. HEK-293T cells were transfected with the Eg.P29 /pEGFP-C1 vector and the corresponding empty vector for 24 hours, and whole cell lysates of different groups were immunoblotted using the specific antibodies. GAPDH was the control protein. ns means no statistical difference, * represents P <0.05, ** represents P <0.01, *** represents P <0.001, and **** represents P <0.0001. The results are from three independent experiments. Error bars represent the standard error of the mean (SEM).

Article Snippet: The membrane was blocked with protein-free rapid blocking buffer (Shanghai Yase Biotechnology Co., Ltd., Shanghai, China) for 15 minutes at room temperature, and the different primary antibodies [β-actin monoclonal antibody, GAPDH monoclonal, Tubulin monoclonal, Vimentin monoclonal antibody, ARPC1A polyclonal antibody, EPLIN (LIMA1) monoclonal antibody, MYH10 monoclonal antibody, Filamin B (FLNB) polyclonal antibody (Proteintech Group, Inc, Wuhan, China), or α-actinin-4 antibody or vinculin antibody (Santa Cruz, Texas, USA)], was added and incubated overnight at 4 °C.

Techniques: Expressing, Transfection, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Screening and identification of protein 29 of Echinococcus granulosus interacting molecules

doi: 10.3389/fcimb.2025.1560436

Figure Lengend Snippet: Verification of Eg.P29-interacting molecules LIMA1 and actin by yeast two-hybrid experiment and co-immunoprecipitation. (A) The self-activation assay of Eg.P29. The bait plasmid Eg.P29 /pGBKT7 and the empty vector pGADT7 were co-transformed into Y2HGold sensory yeast cells for the self-activation assay. The Lam/pGBKT7-T/pGADT7 plasmids were used as a negative control, and the p53/pGBKT7-T/pGADT7 plasmids were used as a positive control. (B) Toxicity assay of Eg.P29. We compared colony growth transformed with the pGBKT7 empty vector and colonies transformed with the Eg.P29 /pGBKT7 vector on an SD/-Trp plate for the toxicity assay. (C) Point-to-point validation of interaction molecules with Eg.P29. The bait plasmid Eg.P29 /pGBKT7 and seven different prey plasmids were co-transformed into Y2HGold yeast cells and coated on SD/-Trp/-Leu/-His/-Ade plates. The interaction relationship of the bait protein and prey proteins was determined by the colony’s growth or not on plates. (D) Spot plate validation of interaction molecules. Selected monoclonal dilutions were cultured on an SD/Leu/-Trp/-His/-Ade/X-α-gal plate for spot plate validation to confirm the interaction of seven protein molecules with Eg.P29 based on whether the colonies’ color changed to blue. (E) Localization of the homologous regions of ACTG1, FLNB, and ARPC1A in the spatial structure of ACTG1. Sequence comparison was performed using MEGA software, the ACTG1 model structure was queried, and homology regions were labeled using the AlphaFold Protein Structure Database. The homologous regions were labeled in green in the figure. (F) Localization of the homologous regions of LIMA1, ACTN4, and MYH10 in the spatial structure of LIMA1. The homologous regions were labeled in green in the figure. Sequence comparison and modeling are the same as (E) . (G) Eg.P29/pCMV-4flag plasmid was transfected into HEK293T cells, and cell lysates were mixed with Anti-flag magnetic beads and then eluted with FLAG peptide to collect the precipitation complexes using a primary antibody to detect interaction molecules by western-blot. GAPDH was the intrinsic reference protein. These experiments were repeated three times.

Article Snippet: The membrane was blocked with protein-free rapid blocking buffer (Shanghai Yase Biotechnology Co., Ltd., Shanghai, China) for 15 minutes at room temperature, and the different primary antibodies [β-actin monoclonal antibody, GAPDH monoclonal, Tubulin monoclonal, Vimentin monoclonal antibody, ARPC1A polyclonal antibody, EPLIN (LIMA1) monoclonal antibody, MYH10 monoclonal antibody, Filamin B (FLNB) polyclonal antibody (Proteintech Group, Inc, Wuhan, China), or α-actinin-4 antibody or vinculin antibody (Santa Cruz, Texas, USA)], was added and incubated overnight at 4 °C.

Techniques: Immunoprecipitation, Activation Assay, Plasmid Preparation, Transformation Assay, Negative Control, Positive Control, Biomarker Discovery, Cell Culture, Sequencing, Comparison, Software, Labeling, Transfection, Magnetic Beads, Western Blot

Journal: Heliyon

Article Title: Filamin B knockdown impairs differentiation and function in mouse pre-osteoblasts via aberrant transcription and alternative splicing

doi: 10.1016/j.heliyon.2024.e39334

Figure Lengend Snippet: Validation of FLNB-regulated genes (DEGs). A) Expression levels of osteogenesis related up-regulated DEG after knockdown of FLNB (FPKM, up) and RT-qPCR measurements (down). B) Expression levels of osteogenesis-associated down-regulated DEG after knockdown of FLNB (FPKM, up) and RT-qPCR measurements (down). C) Protein expression levels of ITGA10, CEBP/β, and GREM1 genes. Compared with sh-NC group, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.

Article Snippet: Then, the membrane was incubated with the appropriate antibody concentration at 4 °C overnight (FLNB, 1:1000, Proteintech; CEBP/β,1:1000, Affitiny; GREM1,1:1000, Affitiny; ITGA10,1:1000, Affitiny; GAPDH,1:5000, Proteintech).

Techniques: Biomarker Discovery, Expressing, Knockdown, Quantitative RT-PCR