Journal: bioRxiv

Article Title: Germline activity of the heat shock factor HSF-1 programs the insulin-receptor daf-2 in C. elegans

doi: 10.1101/2021.02.22.432344

Figure Lengend Snippet: A, Stress resilience of progeny (F1) of day one C. elegans mothers subjected to a short (5 minute) or long (30 minute or 60 minute) heat shock at 34°C. X-axis: the time interval at 20°C after the maternal heat-shock at which progeny were collected. Y axis: Percent F1 progeny that surive severe heat-stress. Legend indicates the duration of maternal heat shock. All the progeny laid by control and heat-shocked mothers within the given time frame were assessed for survival after severe heat stress. n=3-6 experiments/ time point. Each experiment represents 5-15 P0 mothers/ condition/time interval and an average of 24.3±2.3 F1 progeny/time interval/experiment for the short heat shock experiments and 50.4±7.8 F1 progeny/time interval/experiment for longer heat shock experiments. B, Stress resilience of F1 progeny of control mothers (Ctrl), and mothers subjected to the short (5 minute) heat shock following the RNAi mediated downregulation of hsf-1 only in the maternal germline. L4440 empty vector was used as the control. n=4-8 experiments. Each experiment represents thermotolerance of all the progeny laid within the 2-4 hours post-heat shock (average F1 progeny from mothers subjected to germline knockdown using control L4440 RNAi and hsf-1 RNAi: 28.4±1 and 24.1±1.3 /experiment respectively). C, H3K9me2 occupancy (relative to that in control wild-type animals) at the promoter proximal 5’-UTR regions of hsp-70 (C12C8.1)I, hsp-70 (F44E5.4/.5)II and hsp-16.11 V in P0 wild-type animals and hsf-1 (sy441)I animals under control conditions (Ctrl), immediately (HS), and 2 hours after (HS+rec) a 5 minute heat shock (n=4-7 experiments). See and Material and Methods for details regarding the 5’-UTR regions assayed. D, H3K9me2 occupancy (relative to control wild-type animals) at the promoter proximal 5’-UTR regions of hsp-70 (C12C8.1)I, hsp-70 (F44E5.4/.5)II and hsp-16.11 V in P0 animals subjected to RNAi induced kncokdown of met-2 only in the germline. L4440 empty vector was used as the control. H3K9me2 occupancy was assessed in control animals (Ctrl) and at 2 hours recovery post-5’minute heat shock (HS+rec), corresponding to the time point where H3K9me2 levels increase at these regions . (n=3-10 experiments). E, Control and heat-shocked (30 minutes) day one wild-type and hsf-1(sy441) I mutants expressing FLAG::HSF-1 and MET-2::HA were immunoprecipitated with anti-HA antibody. Wild-type animals were also immunoprecipitated using control IgG antibodies. Proteins were separated by SDS/PAGE, and western blot analysis was performed using the antibodies indicated on the left. Representative Western blot shown. 10% of Input is included. (n=3 experiments). F, Representative micrographs showing projections of confocal z-sections through nuclei of oocytes in diakinesis in wild-type animals and hsf-1(sy441)I , under control conditions and upon heat-shock (30 minutes; also see ). Animals express HSF-1 tagged at its endogenous locus with an N-terminal 3X FLAG and MET-2 tagged at its endogenous locus at the C-terminus with 3X HA. Arrows indicate HSF-1 and MET-2 colocalization in nSBs at condensed chromosomes (DAPI) upon heat shock. Panels from left to right show: overlap of HSF-1 immunostaining (red) with DAPI (blue), MET-2 immunostaining alone (green), and overlap of HSF-1 (red) and MET-2 (green) immunostaining. Scale bar:5µm. A-D: Data show Mean ± Standard Error of the Mean *, p <0.05; **, p < 0.01 ***, p <0.001; ns, non-significant; (A-D:: ANOVA with Tukey’s correction; Unpaired Student’s t-test).

Article Snippet: The following secondary antibodies were used: Sheep anti-mouse IgG (H and L) Antibody IRDye 800CW Conjugated (catalog no. 610-631-002, Rockland Immunochemicals) and Alexa Fluor 680 goat anti-rabbit IgG (H+L) (catalog no. A21109, Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot, Immunostaining

Journal: Inflammation Research

Article Title: Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection

doi: 10.1007/s00011-013-0691-3

Figure Lengend Snippet: Working parameters for Western blotting

Article Snippet: Antibodies for CD83 (H-198, SC-20083) and ICAM-1 (H-108, SC-7891) and positive proteins for CD80 (separated Ramos cell) and CD86 (separated Jurkat cell) were purchased from Santa Cruz Biotechnology Inc. Antibodies for CD40 (BWC02, AF632), CD28 (ADS013091, AF-342-PB), CTLA-4 (AF-386-BP), CD80 (AAE02, AF140), CD86 (AAE01, AF-141-NA) and β-actin and standard proteins, namely recombinant human CD80 protein (140-B1) and recombinant human CD86 protein (140-B2), were purchased from R&D Inc. Pre-staining protein marker (P7708S) was obtained from New England BioLabs, (USA), and ABC immunochemical kits from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Western Blot, Autoradiography

Journal: Inflammation Research

Article Title: Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection

doi: 10.1007/s00011-013-0691-3

Figure Lengend Snippet: Quantitative detection of costimulatory proteins or cytokine mRNA in liver. a Identification of CD80 and CD86 in liver. Western blotting was performed using a specific antibody against human costimulatory protein. Recombinant human CD80 and CD86 proteins, positive control CD80 protein (separate Ramos cell) and CD86 protein (separate Jurkat cell), negative control and liver homogenates of human were applied to determination of CD80 and CD86 in liver tissue. In the same western blotting, recombinant protein (Rec), positive control (Pos), negative control (Neg) and liver homogenates of human (Liver) were subjected to electrophoresis. In the photograph of CD86 protein, the molecular weight of recombinant CD86 protein was 90 kDa, and that of CD86 protein in human liver was 80 kDa. In the photograph of CD80, the molecular weight of recombinant CD80 protein was 76 kDa, and that of CD80 protein in human liver was 60 kDa. b Representative western blotting of costimulatory proteins in livers from six separate experiments. Homogenates obtained from the livers in the five groups were subjected to electrophoresis. β-Actin protein served as a protein loading standard. The molecular weight of CD86, CD80, CD83, CD28, CTLA-4, CD40, ICAM-1 and β-actin was 80, 60, 45, 44, 33, 48, 100 and 42 kDa, respectively. The CD80 stains in the ACH or Cir patient were darker than those in the HD, AC or HCC subject. The CD86 stains showed that the stains in the ACH or Cir patient were also darker than those in the HD, AC or HCC subject. In CD83 stains, the photograph showed dark stains of CD83 in the Cir patient. The photograph showed dark stains of CD28 and weak stains of CTLA-4 in the ACH patient. The stains of CTLA-4 in the AC or Cir patient were darker than those in the ACH, HD or HCC patient, whereas the stains in the ACH patient were weaker than those in other subjects. As the CD40 photograph showed, the dark stains of CD40 presented in the ACH or Cir patient. The ICAM-1 stains showed that the stains in the ACH and Cir patients were darker than those in the HD, AC and HCC subjects. c Relative quantity of costimulatory proteins in livers of the five groups ( n = 6, in each group). The images on X-ray membrane were scanned, and the relative quantity of protein was normalized to the protein quantity for each sample using β-actin protein. All the parameters were shown in this figure. The data showed increased CD80, CD86, CD83, CD28 and CD40 and decreased CTLA-4 in the ACH patient, increased CD80, CD86, CD83, CD28, CD40 and CTLA-4 in the Cir patient, increased CTLA-4 and decreased CD80, CD86, CD28 and CD40 in the AC subject, and decreased CD80, CD86, CD28, CD40 and CTLA-4 in the HCC patient. The ICAM-1 levels in the five groups were gradually declined in the order of the ACH group, the Cir group, the HCC group, the AC group and the HD group. d Quantitative detection of cytokines mRNA in liver. The relative quantity of the INFγmRNA, IL-6 mRNA, IL-18 mRNA and IL-10 mRNA in liver in all the five groups was detected by real-time quantitative PCR. The data showed unchanged INFγmRNA, IL-6 mRNA and IL18 mRNA in all the five groups, and increased IL-10 mRNA in the AC or HCC patient

Article Snippet: Antibodies for CD83 (H-198, SC-20083) and ICAM-1 (H-108, SC-7891) and positive proteins for CD80 (separated Ramos cell) and CD86 (separated Jurkat cell) were purchased from Santa Cruz Biotechnology Inc. Antibodies for CD40 (BWC02, AF632), CD28 (ADS013091, AF-342-PB), CTLA-4 (AF-386-BP), CD80 (AAE02, AF140), CD86 (AAE01, AF-141-NA) and β-actin and standard proteins, namely recombinant human CD80 protein (140-B1) and recombinant human CD86 protein (140-B2), were purchased from R&D Inc. Pre-staining protein marker (P7708S) was obtained from New England BioLabs, (USA), and ABC immunochemical kits from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Western Blot, Recombinant, Positive Control, Negative Control, Electrophoresis, Molecular Weight, Real-time Polymerase Chain Reaction

Journal: Inflammation Research

Article Title: Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection

doi: 10.1007/s00011-013-0691-3

Figure Lengend Snippet: Distribution of intrahepatic costimulatory proteins by immunochemical staining. Immunohistochemical staining was performed using a specific antibody against human costimulatory protein. In this figure, seven costimulation stains in liver of the ACH patient were shown. a Distribution of CD80 protein in liver. The hepatocytes were surrounded by CD80 + cells and inflammatory cells, ×400 magnification. b Distribution of CD86. CD86 + cells were localized in the inflammatory zone and they surrounded the hepatocytes, ×400 magnification. c Distribution of CD83 protein by immunochemical stains. CD83 stains mainly appeared in the inflammatory cells, and the CD83 + cells were localized in the inflammatory-necrotic zone, ×400 magnification. d Distribution of CD28 protein in the liver. CD28 + cells were enriched in the inflammatory zone, ×100 magnification. e CTLA-4 distribution in liver. Few CTLA-4 stains were found in the inflammatory zone in the liver of the ACH patient, ×400 magnification. f Distribution CD40 in the liver. CD40 + cells were enriched in the necrotic zone, and CD40 stains surrounded the hepatocytes, ×100 magnification. g ICAM-1 distribution in liver. Most ICAM-1 stains appeared in the hepatic sinus, ×400 magnification

Article Snippet: Antibodies for CD83 (H-198, SC-20083) and ICAM-1 (H-108, SC-7891) and positive proteins for CD80 (separated Ramos cell) and CD86 (separated Jurkat cell) were purchased from Santa Cruz Biotechnology Inc. Antibodies for CD40 (BWC02, AF632), CD28 (ADS013091, AF-342-PB), CTLA-4 (AF-386-BP), CD80 (AAE02, AF140), CD86 (AAE01, AF-141-NA) and β-actin and standard proteins, namely recombinant human CD80 protein (140-B1) and recombinant human CD86 protein (140-B2), were purchased from R&D Inc. Pre-staining protein marker (P7708S) was obtained from New England BioLabs, (USA), and ABC immunochemical kits from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Staining, Immunohistochemical staining

Journal: Inflammation Research

Article Title: Effects of costimulation on intrahepatic immunopathogenesis in patients with chronic HBV infection

doi: 10.1007/s00011-013-0691-3

Figure Lengend Snippet: Pairwise correlation between ALT and costimulatory proteins, or between CTLA-4 and other costimulatory proteins

Article Snippet: Antibodies for CD83 (H-198, SC-20083) and ICAM-1 (H-108, SC-7891) and positive proteins for CD80 (separated Ramos cell) and CD86 (separated Jurkat cell) were purchased from Santa Cruz Biotechnology Inc. Antibodies for CD40 (BWC02, AF632), CD28 (ADS013091, AF-342-PB), CTLA-4 (AF-386-BP), CD80 (AAE02, AF140), CD86 (AAE01, AF-141-NA) and β-actin and standard proteins, namely recombinant human CD80 protein (140-B1) and recombinant human CD86 protein (140-B2), were purchased from R&D Inc. Pre-staining protein marker (P7708S) was obtained from New England BioLabs, (USA), and ABC immunochemical kits from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: