Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Multifunctional Co‐Delivery Systems with Downregulation of the Novel Target PIM1 in Macrophages to Ameliorate TF‐Mediated Coagulopathy in Sepsis

doi: 10.1002/smll.202412688

Figure Lengend Snippet: PIM1 inhibitor SMI‐4a protects septic mice against coagulation activation and sepsis‐induced acute lung injury. A) Schematic representation of animal experimental procedures. B) Representative western blot membranes and corresponding densitometric analyses of (C) PIM1 in lung tissue (n = 6/group). D) mRNA levels of PIM1 in murine lung tissues (n = 5/group). E) Platelet count in mice (n = 4/group). F–H) Plasma levels of coagulation‐related factors in mice plasma by ELISA, including (F) TAT, (G) D‐dimer, and (H) fibrinogen (n = 4/group). I) The lung sections were subjected to hematoxylin and eosin (HE) staining, F4/80, fibrin, and PIM1 immunohistochemical analysis (n = 4/group; scale bar:50 µm). J) Representative western blot membranes and corresponding densitometric analyses of (K) TF, (L) PAI‐1, (M) Thrombin in lung tissue (n = 6/group). N) mRNA levels of TF in murine lung tissues (n = 5/group). O–Q) mRNA levels of (O) IL‐1β, (P) IL‐6, and (Q) TNF‐ɑ in murine lung tissues (n = 5/group). R) Survival curves of mice in all groups (n = 10/group). Each bar represents the mean ± SD. Statistical analysis for three or more groups was carried out using one‐way ANOVA (C‐H and K‐Q). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The concentrations of PIM1 (CSB‐ E11825 h, Cusabio, China) in human plasma and TAT (CSB‐ E08433 m, Cusabio, China), Fbg (CSB‐ E08202 m, Cusabio, China), and D2D (CSB‐ E13584 m, Cusabio, China) in mouse plasma were assessed using ELISA kits according to the guidelines outlined in the respective ELISA kits.

Techniques: Coagulation, Activation Assay, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining

Journal: Nature communications

Article Title: Multiple structures of RNA polymerase II isolated from human nuclei by ChIP-CryoEM analysis.

doi: 10.1038/s41467-025-59580-x

Figure Lengend Snippet: Fig. 2 | Details of the EC-SPT4/5-ELOF1-SPT6 structure. a Domain architectures of the elongation factors. The known domains are indicated. b Close-up view of ELOF1 (purple). c Close-up view around the upstream DNA. d Close-up view of the SPT5 KOW2, KOW3, KOWx, and KOW4 domains. Each KOW domain is indicated with dotted circles. SPT6 is omitted for clarity. e Close-up view of the KOW5 domain. f The SPT6-SPT5 KOW3-RPB4/7 stalk interaction. g The SPT5 KOW1-SPT6

Article Snippet: Themembraneswerewashedwith TBS-T (20mMTris-HCl (pH 7.5), 137mM NaCl, and 0.1% Tween-20) three times and then incubated overnight with anti-FLAG M2 antibody (Sigma # F3165, 1:3000), anti-SPT5 antibody (Proteintech#16511-1-AP, 1:1000), antiSPT6 antibody (Cell Signaling #15616, 1:2000), anti-PAF1 antibody (Cell Signaling #12883, 1:300), anti-RTF1 antibody (Proteintech #12170-1-AP, 1:1000), anti-MED17 antibody (Proteintech#11505-1-AP, 1:2000), or anti-XPB antibody (Cell Signaling #8746, 1:300) in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO #NKB-101).

Techniques:

Journal: Nature communications

Article Title: Decoding the genomic landscape of chromatin-associated biomolecular condensates.

doi: 10.1038/s41467-024-51426-2

Figure Lengend Snippet: Fig. 4 | Experimental validation of potential biomolecular condensates. a Immunofluorescence images of mESC showing that SUPT6H (green) colocalizes with CTR9 (red) and SUPT5H (grey) in puncta. DNA was stained with DAPI (blue). This experimental result was consistent across two independent cell-seeding, fixation, and co-IF staining experiments (each contains three slides). Scale bar: 10 μm. b Line scans of the images of a cell co-stained for SUPT6H, CTR9 and SUPT5H, at the position depicted by the white line. The direction is from the green tick to the purple tick, and the two arrows refer to two representative puncta. FRAP experiments for SUPT6H (c), CTR9 (d) and SUPT5H (e). Left, representative images of the FRAP experiment. The white arrow refers to the punctum undergoing bleaching. Right, quantification of FRAP data for puncta of SUPT6H (n = 6), CTR9 (n = 5) and SUPT5H (n = 5). Puncta were photobleached at t = 0 s, and data were plot

Article Snippet: Following this, the ConA beads bound cells were collected using a magnet and resuspended in 100 μl of antibody buffer containing either 2 μl of DDX21 (Proteintech, 10528-1- AP, lot # 00088037), 4 μl of CTR9 (Bethyl Laboratories, A301-395A, lot # 4), 4 μl of SUPT6H (Novus Biologicals, NB100-2582, lot # 2 A), 1.5 μl of SS18 (Cell Signaling Technology, 21792 (D6I4Z), lot # 1), 1.5 μl of EP300 (Santa Cruz, sc-48343 (F-4), lot # A1323), or 0.5 μl of ELL3 (generously gifted by Prof. Chengqi Lin, Southeast University, China) primary Nature Communications | (2024) 15:6952 13 antibody respectively.

Techniques: Biomarker Discovery, Staining

Journal: Neural regeneration research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury.

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Figure 1 ｜Quantitative changes in thrombin production and cholesterol hydroxylase expression at the lesion site following spinal cord injury (SCI) in a rat model. (A) Experimental design. Experiment I was designed to investigate the effects of the protease-activated receptor 1 (PAR1) inhibitor SCH79797 on thrombin- induced cholesterol-25-hydroxylase (CH25H) expression by astrocytes. For quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, tissue immunofluorescence, and hematoxylin-eosin staining, 1 cm spinal cord segments were harvested from rats at each time point. Experiment II was designed to investigate the effects of rat motor function after SCH79797 treatment. (B) Enzyme-linked immunosorbent assay of thrombin production at 0, 1, 4, and 7 days following SCI. Day 0 was used as the control. (C–E) RNA levels of ch25h, cyp46a1, and cyp27a1 at the lesion site were determined by quantitative polymerase chain reaction at different time points with or without injection of 4.5 μL of 5 mM SCH79797. Quantities were normalized to gapdh levels on day 0. Data are expressed as mean ± SEM (n = 3). *P < 0.05 (independent sample t-test). DMSO: Dimethyl sulfoxide; gapdh: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: After centrifuging at 2750 × g for 5–10 minutes at 2–8°C, the supernatant was subjected to thrombin enzyme-linked immunosorbent assay (ELISA) using a kit (Rat TAT ELISA Kit, Elabscience) according to the manufacturer’s directions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Injection

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Western blot analysis of cell extracts from HEK293 FlpIn cells with tetracycline inducible expression of EGFP-TRIM32 WT , the BBS11 associated mutant EGFP-TRIM32 P130S , or the LGMD2H causing mutant EGFP-TRIM32 D487N shows auto-ubiquitylation activity of TRIM32 WT and TRIM32 P130S , but not the LGMD2H mutant. (B) Sequence of the TRIM32 peptides identified by MS to display PTMs on lysine residues that differed between the TRIM32 proteins with auto-ubiquitylation activity (TRIM32 WT and TRIM32 P130S ) and the LGMD2H disease variant lacking auto-ubiquitylation activity (TRIM32 D487N ). (C) Schematic of TRIM32 domain organization. The D487N mutation in the NHL repeats that is associated with LGMD2H and the P130S mutation in the B-box causing BBS11 are indicated above. The three lysine residues that are focused in this paper are indicated in red, while the PTMs of these residues identified by MS are indicated above. Ub: Ubiquitylation. Ac: Acetylation (D) Western Blot analysis of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , the double mutants EGFP-TRIM32 K247R/K401R , EGFP-TRIM32 K50R/K247R , or EGFP-TRIM32 K247R/K401R , or the triple mutant EGFP-TRIM32 K50R/K247R/K401R . The last lane represents co-transfection of EGFP-TRIM32 WT and mCherry-USP2 expression vectors. The bands representing EGFP-TRIM32, auto-ubiquitylated EGFP-TRIM32, and cleaved EGFP-TRIM32 are indicated to the right. PCNA represents the loading control. (E) Western blot analysis of a similar experiment as in (D), except that the myoblast C2C12 cell line is used instead of the HEK293 FlpIn TRIM32 KO cell line, and the antibody used is an anti-TRIM32 antibody instead of an anti-GFP antibody.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Western Blot, Expressing, Mutagenesis, Activity Assay, Sequencing, Variant Assay, Transfection, Cotransfection, Control

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Schematic of the domain organization of TRIM32 isoform 1 and TRIM32 isoform 3. (B) Western Blot analysis of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 K50R/K247R/K401R , EGFP-TRIM32 K50Q/K247R/K401R , TRIM32 K50R/K247Q/K401R or EGFP-TRIM32 K50R/K247R/K401Q . The bands representing EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Ponceau staining of the membrane represents the loading control. (C) Confocal images of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 K50R/K247R/K401R , EGFP-TRIM32 K50Q/K247R/K401R , TRIM32 K50R/K247Q/K401R or EGFP-TRIM32 K50R/K247R/K401Q , and immunostained with anti-GM130 antibodies, a Golgi marker. The cell nuclei are visualized by DAPI staining. Scale bars: 10 μM.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Western Blot, Transfection, Expressing, Staining, Membrane, Control, Marker

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Confocal images of mCherry-EYFP-TRIM32 WT , mCherry-EYFP-TRIM32 K247R/K401R , mCherry-EYFP-TRIM32 K247Q , mCherry-EYFP-TRIM32 K50R/K247Q/K401R or mCherry-EYFP-TRIM32 K50R/K247R/K401R transiently expressed in HEK293 FlpIn TRIM32 KO cells and exposed to normal or starved conditions. Scale bars: 10 μM. (B) The graphs represent the number of cells with RedOnly dots in the mCherry-EYFP-TRIM32 transfected cells shown with representative images in A. The graphs represent the average of three independent experiments with s.d. (n>20 cells). **P< 0.005; ***P<0.0005 (Student’s t -test).

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Transfection

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Schematic of TRIM32 domain organization with the three regulatory Lysine residues identified in this work indicated above, and the sequence of the mapped PEST sequence indicated below. MutPEST and ΔPEST display the modifications introduced to the predicted PEST sequence. (B) Western Blot analysis of cell extracts from HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 mutPEST , EGFP-TRIM32 ΔPEST , EGFP-TRIM32 K50R/K247R/K401R (3KR), TRIM32 K50R/K247R/K401R/ΔPEST (3KR ΔPEST) or EGFP-TRIM32 K247Q . The bands representing EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Tubulin represents the loading control. (C) Western Blot analysis of cell extracts from HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 WT and mCherry-USP2, EGFP-TRIM32 ΔPEST , EGFP-TRIM32 K50R/K247R/K401R (3KR), TRIM32 K50R/K247R/K401R/ΔPEST (3KR ΔPEST) or EGFP-TRIM32 K50R/247Q/K401R or EGFP-TRIM32 D487N . The bands representing ubiquitylated EGFP-TRIM32, EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Actin represents the loading control.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Sequencing, Western Blot, Transfection, Expressing, Control

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques:

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: Oligonucleotides used in this study.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: