Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A is a novel binding partner for HDAC5. A, FLAG-HDAC5 overexpressed in HEK293T cells was immunopurified using anti-FLAG antibody-agarose resin. The immunoprecipitates were analyzed by SDS-PAGE and silver staining. A high molecular weight protein coimmunoprecipitated with FLAG-HDAC5 and was identified as filamin A by mass spectrometry analysis. IP, immunoprecipitation. B, schematic of HDAC5 constructs. The amino acid numbering refers to human HDAC5. C, filamin A was immunoprecipitated from HEK293T cells transiently expressing the indicated GFP-HDAC5 constructs. The HDAC5 Cterm (residues 1040–1122) is required for HDAC5 interaction with filamin A. D, GFP-Cterm (HDAC51040–1122) was expressed in HEK293T cells and immunoprecipitated using GFP antibody, and the immunoprecipitated material was analyzed by Western blot for GFP and filamin A. TCL, total cell lysate. E, purified GST-Cterm (HDAC51040–1122) was incubated with HEK293T cell lysates, and pulldown material was analyzed by Western blot. Ponceau S staining showed that equal amounts of GST and GST-Cterm were used. The HDAC5 C-terminal domain (residues 1040–1122) is sufficient for HDAC5 interaction with filamin A. F, GFP or GFP-Cterm was overexpressed in HEK293T cells, and endogenous filamin A was immunoprecipitated. Overexpression of GFP-Cterm disrupted the interaction between endogenous filamin A and HDAC5. G, HDAC5 was immunoprecipitated from cultured DRG neurons treated with or without I3A, and immunoprecipitated material was analyzed by Western blot. p-PKC and p-HDAC5 were used as controls for the effectiveness of I3A. H, cultured DRG neurons were treated with the HDAC inhibitor Scriptaid or left untreated, and filamin A was immunoprecipitated. Immunoprecipitated material was analyzed by Western blot for HDAC5 and filamin A. Acetylated histone H3 (Ac-H3) was used as a control for the effectiveness of HDAC inhibition by Scriptaid.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Binding Assay, SDS Page, Silver Staining, High Molecular Weight, Mass Spectrometry, Immunoprecipitation, Construct, Expressing, Western Blot, Purification, Incubation, Staining, Over Expression, Cell Culture, Control, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A is required for HDAC5-mediated tubulin deacetylation. A, cell lysates from filamin A-deficient human melanoma cells (M2) and filamin A-recovered M2 cells (A7) were analyzed for tubulin acetylation by Western blot. The levels of acetylated α-tubulin (Ac-tub) were drastically higher in M2 cells compared with A7 cells, whereas the levels of tyrosinated α-tubulin (Tyr-tub), total α- or β-tubulin, and actin were similar in both cell lines. B, cultured DRG neurons were infected with control shRNA lentivirus or FLNA KD lentivirus and analyzed by Western blot. Filamin A knockdown increased the level of acetylated tubulin in DRG neurons. C, M2 cells expressing GFP-HDAC5 were treated with the PKC activator I3A or left untreated, and the cellular localization of HDAC5 and the level of Ac-tub in the cells was analyzed by immunocytochemistry. I3A-induced cytoplasmic localization of GFP-HDAC5 did not alter Ac-tub levels. Scale bar = 20 μm. D, cell lysates from M2 cells expressing GFP-HDAC5 were analyzed for tubulin acetylation by Western blot. I3A treatment led to the expected phosphorylation of PKCμ and HDAC5 but did not change Ac-tub levels. E, quantification of the Ac-tub levels in C. Data are mean ± S.E.; n = 3; ns, not significant by analysis of variance. F, A7 cells expressing GFP-HDAC5 were treated with I3A alone or with I3A together with the HDAC inhibitor Scriptaid, and the cellular localization of HDAC5 and the levels of Ac-tub in the cells were analyzed by immunocytochemistry. I3A induced translocation of GFP-HDAC5 from the nucleus to the cytoplasm, accompanied by a decrease in the level of Ac-tub. Scriptaid blocked the effect of I3A on the level of Ac-tub without affecting HDAC5 translocation. Scale bar = 20 μm. G, cell lysates from A7 cells expressing GFP-HDAC5 were analyzed for tubulin acetylation by Western blot. I3A treatment induced phosphorylation of PKCμ and HDAC5 and decreased Ac-tub levels. H, quantification of the Ac-tub levels in F. Data are mean ± S.E.; n = 3; **, p < 0.01 by analysis of variance.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Western Blot, Cell Culture, Infection, Control, shRNA, Knockdown, Expressing, Immunocytochemistry, Phospho-proteomics, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A levels increase in axons following injury. A, schematic of the injured sciatic nerve segment used for analysis. B, representative Western blot of control sciatic nerve (−Ax) and sciatic nerve 24 h after axotomy (+Ax). C, quantification of the filamin A levels in A. Data are mean ± S.E.; n = 8; **, p < 0.01 by Student's t test. D, longitudinal sections of a control uninjured nerve and an injured segment of the sciatic nerve within the 3-mm proximal to the injury site, stained for filamin A and the axon marker βIII tubulin (TUJ1 antibody). Scale bar = 100 μm. E, representative image of a 2-mm longitudinal section of injured sciatic nerve stained with filamin A and TUJ1 antibody. Scale bar = 500 μm. Dotted boxes (a and b) indicate the magnified regions 1 mm adjacent to the injury site (a) or 2 mm away from the injury site (b). Scale bar = 50 μm. The dotted arrows indicate the injury site. F, quantification of filamin A fluorescence intensity in unit area a and b in E. AU, arbitrary unit per square micrometer. Data are mean ± S.E. n = 15 for 1 mm and 2 mm from the injury site; ***, p < 0.001 by Student's t test. G, cross-sections of control and injured sciatic nerves stained for filamin A and Schwann cell marker S100β antibodies. Scale bar = 10 μm. H, cross-sections of control and injured sciatic nerves stained with filamin A and TUJ1 antibodies. Scale bar = 10 μm.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Western Blot, Control, Staining, Marker, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A is locally translated in injured axons. A, C, E, and G, Western blots of control (−Ax) and axotomized (+Ax) sciatic nerves treated with vehicle or cycloheximide (CHX, A), EGTA (C), Gö6976 (E), and H-89 (G). B, D, F, and H, quantifications of filamin A levels in A, C, E, and G. Data are mean ± S.E. n = 3; ***, p < 0.001 by Student's t test; ns, not significant.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A is required for HDAC5 localization in growth cones. A, representative images of hippocampal neurons fixed at DIV1 and stained for filamin A, HDAC5, and Ac-tub. Filamin A and HDAC5 colocalized in the growth cone, where the level of acetylated tubulin is low. Scale bar = 5 μm. B, representative images of control or FLNA-KD DRG neurons fixed 3 h after in vitro axotomy and stained for HDAC5 and βIII-tubulin. Knockdown of filamin A reduces HDAC5 levels in the βIII-tubulin-labeled growth cone. Scale bar = 5 μm.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Staining, Control, In Vitro, Knockdown, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A is required for tubulin deacetylation after axon injury. A, control or FLNA-KD DRG neurons were axotomized, fixed 3 h later, and stained for Ac-tub and total α-tubulin. Scale bar = 100 μm. B, average intensity plot of Ac-tub in A. The intensity of Ac-tub was normalized to α-tubulin and plotted in function of distance toward the cell body, with 0 referring to the axotomy site (n = 6 for control and 7 for FLNA KD). Data are mean ± S.E. C, average slopes of the Ac-tub ratio calculated from the plots in B (n = 6 for control and 7 for FLNA KD). Data are mean ± S.E. ***, p < 0.001 by Student's t test. D, control (FUGW lentivirus) or Cterm-overexpressing (FUGW-GFP-Cterm lentivirus) DRG neurons were axotomized, fixed 3 h later, and stained for Ac-tub and total α-tubulin. Scale bar = 100 μm. E, average intensity plot of Ac-tub in D (n = 5 for control and 5 for GFP-Cterm). Data are mean ± S.E. F, average slopes of the Ac-tub ratio calculated from the plots in B (n = 5 for control and 5 for GFP-Cterm). Data are mean ± S.E. ***, p < 0.001 by Student's t test. G, control, FLNA-KD, or GFP-Cterm-overexpressing (FUGW-Cterm lentivirus) DRG neurons were axotomized, fixed 3 h later, and stained for HDAC5 and βIII tubulin (TUJ1 antibody). Scale bar = 100 μm. H, normalized intensity of HDAC5 from G (n = 6, 8, and 8 for control, FLNA KD, and GFP-Cterm, respectively). Data are mean ± S.E. ***, p < 0.001 by analysis of variance).

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

doi: 10.1074/jbc.M115.638445

Figure Lengend Snippet: Filamin A contributes to the regulation of axon regeneration. A, representative images of the in vitro regeneration assay of control or FLNA-KD DRG neurons. Scale bar = 100 μm. B, schematic of the experiment time line. DRG neurons were infected with control or shFLNA lentivirus (shLenti) at DIV3, axotomized at DIV7 to start the in vitro regeneration assay, and stained 40 h later for SCG10. C, average regeneration index obtained from regeneration assays (n = 14 for control and 17 for FLNA KD). Data are mean ± S.E. ***, p < 0.001. D, representative images of in vitro regeneration assay of control (FUGW lentivirus) or GFP-Cterm-overexpressing (FUGW-Cterm lentivirus) DRG neurons. Scale bar = 100 μm. E, schematic of the experiment time line. DRG neurons were infected with control or GFP-Cterm lentivirus at DIV3, axotomized at DIV7 to start the in vitro regeneration assay, and stained 40 h later for SCG10. F, average regeneration index obtained from regeneration assays (n = 8 for each condition). Data are mean ± S.E. **, p < 0.01. G, representative images of the neuron replating assay of control or FLNA-KD DRG neurons. Scale bar = 20 μm. H, schematic of the experiment time line. DRG neurons were infected with control or shFLNA lentivirus at DIV1, replated at DIV4, and fixed at 12 h after replating. I, average of maximum axon length obtained from the neuron replating assays (n = 60 for control and 49 for FLNA KD). Data are mean ± S.E. ***, p < 0.001.

Article Snippet: The filamin A-recovered control cell line A7 was purchased from the ATCC (catalog no. CRL-2500).

Techniques: In Vitro, Control, Infection, Staining

Journal: Biomacromolecules

Article Title: Artificial Protein Cage Delivers Active Protein Cargos to the Cell Interior

doi: 10.1021/acs.biomac.1c00630

Figure Lengend Snippet: Filling and decoration of TRAP-cage. (a) Native PAGE gels showing purified TRAP-cage incubated with His-tagged GFP(-21) after passing through a Ni-NTA column in the absence (−TCEP) or presence (+TCEP) of TCEP. Lane 1: GFP(-21) positive control; 2: molecular weight marker for native PAGE; 3: empty TRAP-cage; 4: input [TRAP-cage with GFP(-21)]; 5 and 8: flow through; 6 and 9: wash; and 7 and 10: elution. Collected fractions were stained for protein (left) or analyzed by fluorescence detection (right, exct. 488 nm). The GFP signal visible in lane 10 of the right-side gel likely reflects GFP still bound to a TRAP ring. (b) Western blot of the gel in (a): collected fractions were subjected to SDS-PAGE followed by western blot with anti-GFP detection. Lane 1: GFP(-21) positive control; 2: molecular weight marker for SDS-PAGE; 3: empty TRAP-cage; 4: input (TRAP-cage with GFP); 5 and 8: flow through; 6 and 9: wash; and 7 and 10: elution. (c) Native PAGE gels showing encapsulation of GFP(-21) by unmodified TRAP-cage or TRAP-cage externally modified with Alexa-647 and PTD4. Lane 1: TRAP-cage with GFP(-21); 2: TRAP-cage with GFP(-21) decorated with Alexa-647; 3: TRAP-cage with GFP(-21) decorated with Alexa-647 and PTD4; and 4: molecular weight marker for native PAGE. Gels were stained for protein (upper panel) and analyzed by fluorescence detection of GFP (middle panel, exct. 488 nm) and Alexa-647 (bottom panel, exct. 647). Gels were imaged using a Biorad Chemidoc detector. (d) Negative stain TEM of TRAP-cage with GFP(-21) (left panel); TRAP-cage with GFP(-21) decorated with Alexa-647 (middle panel); and TRAP-cage with GFP(-21) decorated with Alexa-647 and PTD4 (right panel).

Article Snippet: The membrane was blocked with 5% skimmed milk in Tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T), followed by 1.5 h of incubation with the mouse monoclonal anti-GFP antibody (1:2500; St. John’s Laboratories, UK) and anti-mouse (1:5000, Thermo Fisher Scientific) secondary antibody conjugated with horse radish peroxidase.

Techniques: Clear Native PAGE, Purification, Incubation, Positive Control, Molecular Weight, Marker, Staining, Fluorescence, Western Blot, SDS Page, Encapsulation, Modification

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Detection of K2-acetylated γD-crystallin in the human lens. Western blot analysis of γD-crystallin and N ε -acetyllysine-modified proteins in the human lens. Water-soluble human lens proteins were subjected to Western blot analysis using a monoclonal antibody against γD-crystallin (A). The membrane was stripped and reprobed using a monoclonal antibody against N ε -acetyllysine (B). Densitometry of Western blot B is shown in C. Water-soluble human lens proteins were immunoprecipitated using a monoclonal antibody against γD-crystallin and were subjected to Western blot analysis using an antibody against N ε -acetyllysine (D). The age of the donor lenses is shown below the lanes. M denotes the molecular weight markers. Arrows indicate the positions of the light (LC) and heavy chains (HC) of the antibody. The (−) denotes nonacetylated recombinant γD-crystallin; the (+) denotes in vitro acetylated recombinant γD-crystallin. SDS-PAGE of the purified γD-crystallin is shown in panel E. Lanes 1 and 2 are two preparations of γD-crystallin, and lane 3 is in vitro acetylated γD-crystallin. Western blot analysis of acetylated γD-crystallin using an antibody against N ε -acetyllysine; acetylation was carried out using various molar excess concentrations of Ac 2 O relative to lysine in γD-crystallin (F).

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Western Blot, Modification, Membrane, Immunoprecipitation, Molecular Weight, Recombinant, In Vitro, SDS Page, Purification

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Mass spectrometric detection of acetylation at G1 and K2 in human γD-crystallin. Tandem mass spectra of γD-crystallin from a 73-year-old human lens (A) and in vitro acetylated γD-crystallin (B). The precursor ion of 589.81 (2+) that indicates a mass shift of +84 Da compared with the unmodified peptide is shown. The mass shift of +42 Da was observed at y8, but not y-series ions from y1 to y7, which indicated acetylation of K2. The mass shift of +84 Da was observed on the precursor ion, as well as b-series ions from b2 to b7, which suggested acetylation of K2 and G1.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: In Vitro

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylated γD-crystallin is more prone to thermal aggregation and less protected by α L -crystallin. (A) Time-course aggregation of nonacetylated and acetylated human γD-crystallin at 80 °C. The samples were prepared in 10 mM phosphate buffer containing 1 mM EDTA (pH 7.0). 1: Nonacetylated; 2: acetylated; 3: nonacetylated with 5 mM DTT; and 4: acetylated with 5 mM DTT. The protein concentration was 0.1 mg/mL. (B) Scattering values of both proteins during thermal aggregation after 1 h. (C) Time-course aggregation of nonacetylated and acetylated γD-crystallin at 80 °C in the presence/absence of α L -crystallin. The samples were prepared in 10 mM phosphate buffer containing 5 mM DTT and 1 mM EDTA (pH 7). The concentration of both γD-crystallin proteins was 0.1 mg/mL. 1: Nonacetylated; 2: acetylated; 3: α L -crystallin alone; 4: nonacetylated + α L -crystallin (1:1) (w/w); 5: acetylated + α L -crystallin (1:1) (w/w); 6: nonacetylated + α L -crystallin (1:0.75) (w/w); and 7: acetylated + α L -crystallin (1:0.75)(w/w). (D) Percent protection of nonacetylated and acetylated γD-crystallin by α L -crystallin during thermal aggregation after 1 h. Bars represent the means ± SD of three independent experiments. * p < 0.05 and *** p < 0.0005.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Protein Concentration, Concentration Assay

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylation perturbed only the tertiary structure of γD-crystallin. (A) Far-UV CD spectra of nonacetylated and acetylated human γD-crystallin. (B) Near-UV CD spectra of nonacetylated and acetylated human γD-crystallin. The concentrations of the protein samples used in far- and near-UV CD were 0.2 and 1.0 mg/mL, respectively. (C) Intrinsic tryptophan fluorescence spectra of nonacetylated and acetylated human γD-crystallin (0.025 mg/mL) were recorded from 310 to 400 nm. The excitation wavelength was 295 nm. Excitation and emission slit widths were 5 nm each. The data were collected at a 0.5 nm wavelength resolution. All assays were performed in 10 mM phosphate buffer containing 1 mM EDTA and 5 mM DTT (pH 7.0) at 25 °C.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Circular Dichroism, Fluorescence

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Conformational analysis of nonacetylated and acetylated γD-crystallin using molecular dynamics simulations. Molecular dynamics simulation results suggest that acetylation does not alter the secondary structure of γD-crystallin (A). Tryptophan residues in the nonacetylated (yellow), in the K2-acetylated (orange) and in the G1- and K2-acetylated γD-crystallin (blue) models are shown (B). The structures used for superposition are models from the top conformational cluster. All tryptophan residues were found to be buried within the globular core of the proteins. The “circled” portions in the panel B represents modulation in the structure/conformation of human γD-crystallin due to aceylation.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylation alters the cysteine microenvironment in γD-crystallin. DTNB reaction kinetic profiles of nonacetylated and acetylated γD-crystallin at 25 °C. A protein concentration of 0.1 mg/mL in 50 mM phosphate buffer containing 1 mM EDTA (pH 7.4) was used. DTNB was used at a 7-fold molar excess of the protein. Absorbance was measured at 412 nm as a function of time. Mycobacterium leprae HSP18, which lacks cysteine residues, was used as a negative control.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Protein Concentration, Negative Control

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Orientation of thiol residues in nonacetylated and acetylated γD-crystallin. The relative distance between C18 and C78 in the nonacetylated (A), K2-acetylated (B), and G1- and K2-acetylated γD-crystallin (C) calculated from the top conformational cluster models. Heat map analysis for C18 and C78 of human γD-crystallin [nonacetylated (D), K2-acetylated (E), and G1- and K2-acetylated γD-crystallin (F)] was prepared using the root-mean-square deviation of C18 and C78 as collective variables for the y -axis and the distance between C18 and C78 as the collective variable for the x -axis.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Surface electrostatic potential attributable to acetylation in human γD-crystallin. Molecular electrostatic potential surfaces for nonacetylated (A), K2-acetylated (D), and G1- and K2-acetylated (G) human γD-crystallin that were obtained using the adaptive Poisson–Boltzmann solver. Blue and red contours represent electropositive and electronegative isosurfaces at ±0.3 kT/e, respectively. The residues within a 4-Å radius of K2 (green) and G1 (cyan) are represented as sticks with hydrogens, and hydrogen bonds are represented as red dotted lines (B, E, and H). The vacuum-generated electrostatic potentials near the acetylation region are highlighted in all models, and the models show the electropositive (nonacetylated: C) and electronegative potentials (K2 acetylation: F; G1 and K2 acetylation: I). The yellow arrow in panel D, F, G, and I depicts comparative changes due to acetylation of human γD-crystallin.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Generated

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylated γD-crystallin is structurally less stable than the nonacetylated γD-crystallin. Equilibrium GdnHCl unfolding profile for 0.025 mg/mL of nonacetylated and acetylated human γD-crystallin at 37 °C (A). The profile was normalized to a scale of 0–1. Symbols represent the experimental data points, and the solid lines represent the best fit according to the three-state model. The thermal stability of both proteins was evaluated by far-UV CD measurements (B). The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2 min equilibration. A protein concentration of 0.1 mg/mL in 10 mM phosphate buffer, 5 mM DTT, and 1 mM EDTA (pH 7.0) was used. The raw data were fitted to a two-state model, and the fitting results are represented by solid lines. The thermal stability of both proteins was evaluated by monitoring the intrinsic tryptophan fluorescence (C). The temperature was controlled by a water bath, and the data were recorded at a given temperature after a 2 min equilibration. A protein concentration of 0.025 mg/mL in 10 mM phosphate buffer, 5 mM DTT, and 1 mM EDTA (pH 7.0) was used. The raw data were fitted to a two-state model, and the fitting results are represented by solid lines. Potential energy estimation computed from the molecular dynamics simulation (D). The relative difference in the potential energy profiles indicate the stability of the macro-models in the order nonacetylated > K2-acetylated > G1- and K2-acetylated.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Protein Concentration, Fluorescence

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylation alters the unfolding and refolding of γD-crystallin. Productive kinetic unfolding data of nonacetylated and acetylated human γD-crystallin (A). For unfolding, nonacetylated proteins (0.1 mg/mL) were diluted into 5.5 M GdnHCl, 10 mM phosphate buffer, 5 mM DTT, and 1 mM EDTA (pH 7.0) at 25 °C. Changes in the fluorescence intensity at 355 nm were monitored over time using an excitation wavelength of 295 nm. The final protein concentration in the unfolding buffer was 0.01 mg/mL. Unfolding time-course profiles (B) of both proteins were fitted with double and single exponentials, respectively, as indicated by solid lines. Productive kinetic refolding data of nonacetylated and acetylated human γD-crystallin (C). The protein (0.1 mg/mL) was initially unfolded in 5.5 M GdnHCl and diluted into 10 mM phosphate, 5 mM DTT, and 1 mM EDTA (pH 7.0) at 25 °C to yield a final GdnHCl concentration of 1.0 M. The final protein concentration was 0.01 mg/mL. Changes in the fluorescence intensity at 355 nm were monitored over time using an excitation wavelength of 295 nm. Both refolding time-course profiles were fitted with double exponentials, as indicated by solid lines (D).

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Fluorescence, Protein Concentration, Concentration Assay

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Acetylation exposes additional hydrophobic sites at the surface of γD-crystallin. The concentrations of the protein samples and bis-ANS were 2.5 μM and 10 μM, respectively. All samples were prepared in 10 mM phosphate buffer, 5 mM DTT, and 1 mM EDTA (pH 7.0). The fluorescence spectrum of bis-ANS bound to different samples was recorded from 450 to 600 nm at 25 °C. The excitation wavelength was 390 nm.

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Fluorescence

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Detection of Acetylated Amino Acids in Human γD-Crystallin

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques: Sequencing, Modification, In Vitro, In Vivo

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: C 1/2 and Δ G 0 Values of Nonacetylated and Acetylated Human γD-Crystallin at 37 °C

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Thermal Unfolding Parameters of Nonacetylated and Acetylated Human γD-Crystallin Determined from Far-UV CD Spectroscopy

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Thermal Unfolding Parameters of Nonacetylated and Acetylated Human γD-Crystallin Determined from the Intrinsic Tryptophan Fluorescence

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Productive Kinetic Unfolding Parameters of Nonacetylated and Acetylated Human γD-Crystallin

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Biochemistry

Article Title: Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

doi: 10.1021/bi501004y

Figure Lengend Snippet: Productive Kinetic Refolding Parameters of Nonacetylated and Acetylated Human γD-Crystallin

Article Snippet: Water-soluble proteins (20 μg from each lens) were analyzed on a 12% denaturing gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against human γD-crystallin (1:2500 dilution, Santa Cruz Biotechnology, Dallas, TX) and an HRP-conjugated goat antimouse IgG (1:5000 dilution, Promega, Madison, WI).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

doi: 10.1074/jbc.m109.029728

Figure Lengend Snippet: FIGURE 3. Quantification of the total protein expression levels of alanine mutantsofHgt1pshowingsevereeffectonactivity(GroupImutants)(A) and moderate effect on activity (Group II) (B). The crude extracts prepared from the met15hgt1 strain (ABC 817) transformed with plasmids bearing the different alanine mutants of Hgt1p were prepared, and 20 g of total protein was resolved using 9% SDS-PAGE, electroblotted to a nitrocellulose membrane. The blot was probed with mouse anti-HA (Cell Signaling) at 1:1000 dilution as primary antibody and goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Cell Signaling, at 1:2500 dilution) as sec- ondary antibody. The signal was detected using ECL kit (Amersham Bio- sciences). Molecular weight markers (SM0431, MBI Fermentas) were used to estimate the protein molecular weight. The total protein was quantified by densitometryanalysisofproteinbands.Thedataareexpressedaspercentage protein expression normalized to the wild-type (WT) expression level and are the mean of the protein expression levels obtained in three independent experiments. A representative blot is shown in the inset. Equal loading of the proteins (20 g) in each well of the gel was visually monitored by Coomassie staining of a duplicate gel and Ponceau S staining of the membrane after transfer (data not shown).

Article Snippet: The blot was probed with mouse anti-HA (Cell Signaling) at 1:1000 dilution as primary antibody and goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Cell Signaling, at 1:2500 dilution) as secondary antibody.

Techniques: Expressing, Activity Assay, Transformation Assay, SDS Page, Membrane, Molecular Weight, Staining

Journal: Journal of Biological Chemistry

Article Title: Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

doi: 10.1074/jbc.m109.029728

Figure Lengend Snippet: FIGURE 4. Cell surface localization of alanine mutants of Hgt1p show- ing a severe effect on activity (Group I mutants) and moderate effect on activity (Group II). The strain met15hgt1 (ABC 817) was trans- formed with plasmids bearing the different alanine mutants of Hgt1p and labeled by indirect immunofluorescence using mouse anti-HA (Cell Sig- naling) primary antibody (at 1:100 dilution) and goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Cell Signaling) secondary antibody (at 1:500 dilution) and visualized using confocal microscope, as described under “Experimental Procedures.” Only fluorescence images have been shown. WT, wild type.

Article Snippet: The blot was probed with mouse anti-HA (Cell Signaling) at 1:1000 dilution as primary antibody and goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Cell Signaling, at 1:2500 dilution) as secondary antibody.

Techniques: Activity Assay, Labeling, Immunofluorescence, Microscopy, Fluorescence

Journal: Journal of cell science

Article Title: RPA facilitates rescue of keratinocytes from UVB radiation damage through insulin-like growth factor-I signalling.

doi: 10.1242/jcs.255786

Figure Lengend Snippet: Fig. 1. Effect of inhibitors on IGF-I mediated rescue in primary monolayer keratinocytes. Keratinocytes were treated with inhibitors for specific time periods before UVBR (100 mJ/cm2) and 20 ng/ml IGF-I treatment. (A) Cell survival was assessed using an MTT assay. (B) Apoptosis was visualised using Annexin-V FITC/propidium iodide staining under a fluorescence microscope and quantified. (C) CPDs were detected using anti-thymidine dimer immunostaining under a fluorescence microscope. Scale bars: 50 µm. (D) Treated cells were fixed, stained with propidium iodide and the number of cells in each phase of the cell cycle was analysed using a flow cytometer. (E) Cells were then lysed and protein was separated by SDS-PAGE, following which they were transferred to a nitrocellulose membrane and probed to determine the relative abundance of key signalling mediators of the IGF-1R signalling cascade. The molecular weights of the proteins were as follows: IGF-1R, 95 kDa; Akt, 60 kDa; ERK1/2, 42 kDa and 44 kDa, respectively; p53, 53 kDa; p21, 21 kDa; FAK, 125 kDa; and GAPDH, 36 kDa. GAPDH served as a loading control and the representative blots are from a single experiment. Band intensity was first normalised to the loading control and then to total protein levels. Experiments were performed in keratinocytes from three independent skin donors (n=3). Data are mean±s.e.m. *P≤0.05, **P≤0.01, ***P≤0.001 [one-way ANOVA and Tukey’s multiple comparisons test as compared to the post-UVBR IGF-I-treated keratinocytes (without inhibitors)].

Article Snippet: Primary antibodies used were as follows: Akt (1:1000 dilution, Cell Signaling Technology); anti-Akt mouse monoclonal (2H10, 2967, 1:1000 dilution); anti-phospho Akt rabbit monoclonal (Ser 473) (193H12, 4058, Cell Signaling Technology); ERK1/2 [1:2500 dilution, anti-ERK 1/2 rabbit polyclonal (p42/44), 9102, and anti-phospho ERK1/2 MAPK mouse monoclonal, 1:2500 dilution, Thr 202/Tyr 204 (E10), 9106, Cell Signaling Technology]; IGF-IR [1:1000 dilution, anti-IGF-1R mouse monoclonal, 3027, and 1:500 dilution, anti-phospho IGF-1R rabbit monoclonal (Tyr 1135/1136) (19H7), 3024, Cell Signaling Technology]; p21 [1:2500 dilution, anti-p21 Waf1/Cip1 (12D1) rabbit monoclonal, 2947, Cell Signaling Technology]; p53 (1:2500 dilution, anti-p53 antibody, 9282, Cell Signaling Technology); FAK [1:2500 dilution; anti-FAK rabbit polyclonal, 3285, Cell Signaling Technology, and 1:1000 dilution, antiphospho FAK (Tyr397) antibody, clone 18, 05-1140, Merck Millipore, USA]; and RPA-1 [1:2500 dilution, anti-RPA 70 kDa subunit (H7), sc48425, Santa Cruz Biotechnology].

Techniques: MTT Assay, Staining, Fluorescence, Microscopy, Immunostaining, Flow Cytometry, SDS Page, Membrane, Control

Journal: Journal of cell science

Article Title: RPA facilitates rescue of keratinocytes from UVB radiation damage through insulin-like growth factor-I signalling.

doi: 10.1242/jcs.255786

Figure Lengend Snippet: Fig. 2. Effect of inhibitors on IGF-I-mediated rescue in primary keratinocyte co-cultures. Keratinocytes in co-culture with fibroblasts were treated with inhibitors for specific time periods before UVBR (100 mJ/cm2) and 20 ng/ml IGF-I treatment. (A) Cell survival was assessed using an MTT assay. (B) Apoptosis was visualised using Annexin-V FITC/PI staining under a fluorescence microscope and quantified. (C) CPDs were detected using anti-thymidine dimer immunostaining under a fluorescent microscope. Scale bars: 50 µm. (D) Treated cells were fixed, stained with propidium iodide and the number of cells in each phase of the cell cycle was analysed using a flow cytometer. (E) Cells were then lysed and protein was separated by SDS-PAGE, following which they were transferred to a nitrocellulose membrane and probed to determine the relative abundance of key signalling mediators of the IGF-1R signalling cascade. The molecular weights of the proteins were as follows: IGF-1R, 95 kDa; Akt, 60 kDa; ERK1/2, 42 kDa and 44 kDa, respectively; p53, 53 kDa; p21, 21 kDa; FAK, 125 kDa; and GAPDH, 36 kDa. GAPDH served as a loading control and the representative blots are from a single experiment. Band intensity was first normalised to the loading control and then to total protein levels. Experiments were performed in keratinocytes from three independent skin donors (n=3). Data are mean±s.e.m. *P≤0.05, **P≤0.01, ***P≤0.001 [one-way ANOVA and Tukey’s multiple comparisons test as compared to the post-UVBR IGF-I-treated keratinocytes (without inhibitors)].

Article Snippet: Primary antibodies used were as follows: Akt (1:1000 dilution, Cell Signaling Technology); anti-Akt mouse monoclonal (2H10, 2967, 1:1000 dilution); anti-phospho Akt rabbit monoclonal (Ser 473) (193H12, 4058, Cell Signaling Technology); ERK1/2 [1:2500 dilution, anti-ERK 1/2 rabbit polyclonal (p42/44), 9102, and anti-phospho ERK1/2 MAPK mouse monoclonal, 1:2500 dilution, Thr 202/Tyr 204 (E10), 9106, Cell Signaling Technology]; IGF-IR [1:1000 dilution, anti-IGF-1R mouse monoclonal, 3027, and 1:500 dilution, anti-phospho IGF-1R rabbit monoclonal (Tyr 1135/1136) (19H7), 3024, Cell Signaling Technology]; p21 [1:2500 dilution, anti-p21 Waf1/Cip1 (12D1) rabbit monoclonal, 2947, Cell Signaling Technology]; p53 (1:2500 dilution, anti-p53 antibody, 9282, Cell Signaling Technology); FAK [1:2500 dilution; anti-FAK rabbit polyclonal, 3285, Cell Signaling Technology, and 1:1000 dilution, antiphospho FAK (Tyr397) antibody, clone 18, 05-1140, Merck Millipore, USA]; and RPA-1 [1:2500 dilution, anti-RPA 70 kDa subunit (H7), sc48425, Santa Cruz Biotechnology].

Techniques: Co-Culture Assay, MTT Assay, Staining, Fluorescence, Microscopy, Immunostaining, Flow Cytometry, SDS Page, Membrane, Control

Journal: Journal of cell science

Article Title: RPA facilitates rescue of keratinocytes from UVB radiation damage through insulin-like growth factor-I signalling.

doi: 10.1242/jcs.255786

Figure Lengend Snippet: Fig. 3. Effect of inhibitors on IGF-I-mediated rescue in primary keratinocytes of HSEs. Primary keratinocytes and fibroblasts were seeded on de-epidermised dermis, cultured at the air-liquid interface and exposed to 50 mJ/cm2 UVBR in the presence of inhibitors and 20 ng/ml IGF-I for specific time periods. (A) Histological analysis was performed by H&E staining and ‘sunburnt’ cells were quantified. Scale bars: 50 µm. (B) Apoptosis in primary keratinocytes present in epidermis of HSEs was assessed using immunohistochemistry for cleaved caspase-3, visualised using a light microscope and quantified using a semi- automated technique using Image J. (C) Quantification of CPD in primary keratinocytes present in epidermis of HSEs was assessed using immunohistochemistry against anti-thymidine dimer. The CPD positive cells were visualised using a light microscope and quantified using a semi-automated technique using Image J software. (D) Once isolated, the cells were fixed, stained with propidium iodide and the number of cells in each phase of the cell cycle was analysed using a flow cytometer. (E) Cells were isolated from the epidermis and lysed, and protein was separated by SDS-PAGE, following which they were transferred to a nitrocellulose membrane and probed to determine the relative abundance of key signalling mediators of the IGF-1R signalling cascade. The molecular weight of the proteins were as follows: IGF-1R, 95 kDa; Akt, 60 kDa; ERK1/2, 42 kDa and 44 kDa, respectively; p53, 53 kDa; p21, 21 kDa; FAK, 125 kDa; and GAPDH, 36 kDa. GAPDH served as a loading control and the representative blots are from a single experiment. Band intensity was first normalised to the loading control and then to total protein levels. Experiments were performed in keratinocytes from three independent skin donors (n=3). Data are mean±s.e.m. *P≤0.05, **P≤0.01, ***P≤0.001 [one-way ANOVA and Tukey’s multiple comparisons test as compared to the post-UVBR IGF-I-treated keratinocytes (without inhibitors)].

Article Snippet: Primary antibodies used were as follows: Akt (1:1000 dilution, Cell Signaling Technology); anti-Akt mouse monoclonal (2H10, 2967, 1:1000 dilution); anti-phospho Akt rabbit monoclonal (Ser 473) (193H12, 4058, Cell Signaling Technology); ERK1/2 [1:2500 dilution, anti-ERK 1/2 rabbit polyclonal (p42/44), 9102, and anti-phospho ERK1/2 MAPK mouse monoclonal, 1:2500 dilution, Thr 202/Tyr 204 (E10), 9106, Cell Signaling Technology]; IGF-IR [1:1000 dilution, anti-IGF-1R mouse monoclonal, 3027, and 1:500 dilution, anti-phospho IGF-1R rabbit monoclonal (Tyr 1135/1136) (19H7), 3024, Cell Signaling Technology]; p21 [1:2500 dilution, anti-p21 Waf1/Cip1 (12D1) rabbit monoclonal, 2947, Cell Signaling Technology]; p53 (1:2500 dilution, anti-p53 antibody, 9282, Cell Signaling Technology); FAK [1:2500 dilution; anti-FAK rabbit polyclonal, 3285, Cell Signaling Technology, and 1:1000 dilution, antiphospho FAK (Tyr397) antibody, clone 18, 05-1140, Merck Millipore, USA]; and RPA-1 [1:2500 dilution, anti-RPA 70 kDa subunit (H7), sc48425, Santa Cruz Biotechnology].

Techniques: Cell Culture, Staining, Immunohistochemistry, Light Microscopy, Software, Isolation, Flow Cytometry, SDS Page, Membrane, Molecular Weight, Control

Journal: Journal of cell science

Article Title: RPA facilitates rescue of keratinocytes from UVB radiation damage through insulin-like growth factor-I signalling.

doi: 10.1242/jcs.255786

Figure Lengend Snippet: Fig. 4. Effect of RPA knockdown on IGF-I-mediated keratinocyte rescue. Primary keratinocytes in monolayers (i) and co-culture with fibroblasts (ii) were transfected for RPA knockdown and cultured in the presence or absence of 20 ng/ml IGF-I for 1 h post-UVBR treatment (100 mJ/cm2). (A) Cell survival was assessed using an MTT assay. (B) Apoptosis was visualised using Annexin-V FITC/PI staining under a fluorescence microscope and quantified. (C) CPDs were detected using anti-thymidine dimer immunostaining under a fluorescence microscope. Scale bars: 50 µM. (D) Treated cells were fixed, stained with propidium iodide and the number of cells in each phase of the cell cycle was analysed using a flow cytometer. (E) Cells were lysed and protein was separated by SDS-PAGE, following which they were transferred to a nitrocellulose membrane and probed to determine the relative abundance of key signalling mediators of the IGF-1R signalling cascade. The molecular weights of the proteins were as follows: IGF-1R, 95 kDa; Akt, 60 kDa; ERK1/2, 42 kDa and 44 kDa, respectively; p53, 53 kDa; p21, 21 kDa; and GAPDH, 36 kDa. GAPDH served as a loading control and the representative blots are from a single experiment. Band intensity was first normalised to the loading control and then to total protein levels. Experiments were performed in keratinocytes from three independent skin donors (n=3). Data are mean±s.e.m. *P≤0.05, **P≤0.01, ***P≤0.001 (one-way ANOVA and Tukey’s multiple comparisons test as compared to the no siRNA post-UVBR IGF-I-treated keratinocytes). UR represents the unirradiated cells, IR represents the irradiated cells and IGF-I represents the post-UVBR IGF-I-treated cells. The presence or absence of siRNA-mediated knockdown is represented by siRNA.

Article Snippet: Primary antibodies used were as follows: Akt (1:1000 dilution, Cell Signaling Technology); anti-Akt mouse monoclonal (2H10, 2967, 1:1000 dilution); anti-phospho Akt rabbit monoclonal (Ser 473) (193H12, 4058, Cell Signaling Technology); ERK1/2 [1:2500 dilution, anti-ERK 1/2 rabbit polyclonal (p42/44), 9102, and anti-phospho ERK1/2 MAPK mouse monoclonal, 1:2500 dilution, Thr 202/Tyr 204 (E10), 9106, Cell Signaling Technology]; IGF-IR [1:1000 dilution, anti-IGF-1R mouse monoclonal, 3027, and 1:500 dilution, anti-phospho IGF-1R rabbit monoclonal (Tyr 1135/1136) (19H7), 3024, Cell Signaling Technology]; p21 [1:2500 dilution, anti-p21 Waf1/Cip1 (12D1) rabbit monoclonal, 2947, Cell Signaling Technology]; p53 (1:2500 dilution, anti-p53 antibody, 9282, Cell Signaling Technology); FAK [1:2500 dilution; anti-FAK rabbit polyclonal, 3285, Cell Signaling Technology, and 1:1000 dilution, antiphospho FAK (Tyr397) antibody, clone 18, 05-1140, Merck Millipore, USA]; and RPA-1 [1:2500 dilution, anti-RPA 70 kDa subunit (H7), sc48425, Santa Cruz Biotechnology].

Techniques: Knockdown, Co-Culture Assay, Transfection, Cell Culture, MTT Assay, Staining, Fluorescence, Microscopy, Immunostaining, Flow Cytometry, SDS Page, Membrane, Control, Irradiation

Journal: Molecular Psychiatry

Article Title: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior

doi: 10.1038/s41380-018-0260-9

Figure Lengend Snippet: Molecular identity of SERT+ neurons in the prefrontal cortex (PFC). a Transient SERT expression in the PFC during postnatal development revealed by in situ hybridization on coronal sections through the frontal pole (postnatal ages (P): 4, 7, 10, and 14). b Immunolabeling against Ctip2 (layer 5), Foxp2 (layer 6), and GFP (SERT Cre/+ ) in the PFC. b’ SERT-GFP neurons often colocalize with Ctip2 (arrowheads), Foxp2 (white arrows), or both (yellow arrows). c GFP-expressing neurons from the PFC of SERT Cre/+ :RCE mice were dissected and subsequently isolated using FACS (in c' , individual cells indicated by arrows). d Expression levels of monoamine-related transcripts in isolated PFC SERT-GFP neurons from ( c' ) represented by the normalized read counts obtained after deep transcriptome sequencing. e Expression levels of cortical layer-specific molecular markers after transcriptome analysis indicating an enrichment of deep layer markers (layers 5 and 6) in PFC SERT-GFP neurons

Article Snippet: The following primary antisera were used: anti-5-HT (rabbit from Sigma, S5545, 1/5000; goat from Abcam, 66047, 1/1000), anti-GFP (rabbit, Molecular Probes, A6455, 1/2500; chicken, Aves, GFP-1020, 1/1000), anti-DsRed (rabbit, Clontech, 632496, 1:400), anti-Ctip2 (rat, Abcam, ab18465, 1/500), anti-Cux1 (rabbit, Santa Cruz Biotechnology, sc-13024, 1/1000), anti-Foxp2 (goat, Santa Cruz Biotechnology, sc-21069, 1/500), and anti-cfos (rabbit, Abcam, 190289, 1/1000).

Techniques: Expressing, In Situ Hybridization, Immunolabeling, Isolation, Sequencing

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A-B) Confocal images of YFP-PrP* NRK cells at steady state. Scale bar represents 10 µm. (A) Immunofluorescence image of endogenous calnexin (CNX) in a YFP-PrP* NRK cell. The nucleus was stained with Hoechst. (B) Immunofluorescence image of cell-surface YFP-PrP* using anti-GFP antibody on cells that were not permeabilized. The nucleus was stained with Hoechst. (C-D) Confocal images of GFP-CD59 (C94S) NRK cells at steady state. Scale bar represents 10 µm. (C) Immunofluorescence image against endogenous calnexin (CNX) in GFP-CD59 (C94S). The nucleus was stained with Hoechst. (D) Immunofluorescence of cell-surface GFP-CD59 (C94S) using anti-GFP antibody on cells that were not permeabilized. The nucleus was stained with Hoechst. (E) Western blots of GFP-tag co-immunoprecipitates from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells at steady state. Blots were probed for GFP for YFP-PrP*, and probed for endogenous calnexin (CNX), TMP21, TMED2 and TMED9. (F) Western blots of GFP-tag co-immunoprecipitates from parental untransfected NRK cells (P) or stably transfected GFP-CD59 (C94S) NRK cells (n=1). Blots were probed for GFP for GFP-CD59 (C94S), and probed for endogenous calnexin (CNX), TMP21, TMED2 and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Immunofluorescence, Staining, Western Blot, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Time-lapse imaging sequence collected immediately after thapsigargin (TG)-treatment of a typical YFP-PrP* NRK cell that was co-expressing ER-marker, Cerulean-calnexin (CER-CNX). Scale bar represents 10 µm. (B) Plot of the average Pearson’s r values between YFP-PrP* and CER-CNX for 9 individual cells across the 0 and 20 min time points of time-lapses collected after addition of TG, as shown in (A). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CER-CNX within the boundaries of the cell. For each image, the boundary of the cell was revealed by temporarily increasing the gain for CER-CNX. The Pearson’s r value for individual cells are color-coded. (C) Time-lapse imaging sequence collected immediately after TG-treatment of a typical YFP-PrP* NRK cell that was co-expressing the Golgi marker, CER-GalT. Scale bar represents 10 µm. (D) Plot of the average Pearson’s r values between YFP-PrP* and CER-GalT for 10 individual cells across the T0 and T20min time points of time-lapses collected after addition of TG, as shown in (C). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CER-GalT within the boundaries of the cell. For each image, the boundary of the cell was revealed by temporarily maximizing the gain for YFP-PrP*. The Pearson’s r value for individual cells are color-coded. (E) Time-lapse images collected immediately after TG-treatment of a typical YFP-PrP* NRK cell that was co-expressing CER-TMED9 and FusionRed (FusRed)-SiT as a Golgi-marker. This figure panel is also provided as Video 1. Scale bar represents 10 µm. (F) Plot of the average Pearson’s r values between YFP-PrP* and FusRed-SiT or CER-TMED9 and FusRed-SiT, as indicated, for 6 individual cells across the 0 and 20min time points of time-lapses collected after addition of TG, as shown in (E). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and FusRed-SiT or CER-TMED9 and FusRed-SiT, within the boundaries of the cell. For each data point at 0 and 20 min time points, the boundary of the cell was revealed by temporarily maximizing the gain for YFP-PrP*. The Pearson’s r value for individual cells are color-coded.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Imaging, Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of GFP-tag co-immunoprecipitations from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells (n=3 biological replicates). YFP-PrP* NRK cells were treated with thapsigargin (TG) and collected for co-immunoprecipitation at the indicated time points. In addition to using anti-GFP antibody to detect co-immunoprecipitation of YFP-PrP*, blots were probed for calnexin (CNX), TMED9 and TMP21. “L” indicates the lane that the ladder was loaded in. (B) Bar graph representing the mean band intensity for CNX, TMED9 and TMP21 in the eluates from 3 independently performed experiments as shown in (A). For each time point, CNX, TMED9 and TMP21 eluate band intensities were double normalized. First, they were normalized by the band intensity of eluate YFP-PrP*. Second, they were normalized against eluate time 0 band intensity of each protein probed. Error bars represent standard deviations of the mean of 3 independent experiments. Symbols were coded for each experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** indicated p<0.01.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of control (siTMED9 –) or siTMED9-treated (siTMED9 +) YFP-PrP* NRK cells that were either untreated (TG –) or treated with TG for 90 min (TG +) (n=3 biological replicates). Blots were probed for TMED9, PrP to detect YFP-PrP* and GAPDH. Each lane was numbered to facilitate cross-referencing with the quantification shown in (B). (B) Bar graph representing the mean band intensity for YFP-PrP* from 3 biological replicates as shown in (A). For each condition, YFP-PrP* band intensities were double normalized. First, they were normalized by the band intensity of GAPDH. Second, they were normalized against the untreated control (“siTMED9 – TG – “) band intensity. Error bars represent standard deviation of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** indicated p<0.01. The bars were numbered 1-4 to facilitate cross-referencing with the representative blot in (A). (C) Time-lapse images of control (CTL) or TMED9 siRNA (siTMED9)-treated YFP-PrP* NRK cells. Prior to starting the time-lapses, nuclei were stained with Hoechst. Image-collection was started immediately after the addition of thapsigargin (TG). Scale bar represents 20 µm. (D) Percentage of cells as represented in (C) with a perinuclear Golgi-localized pattern of YFP-PrP* after 30 min of TG treatment. The bar graph represents 3 biological replicates. Symbols were coded for each independently executed experiment. The number of cells analyzed for each biological replicate are as follows (CTL: triangle 29, diamond 56, circle 51; siTMED9: triangle 69, diamond 58, circle 61). Error bars represent standard deviation of the mean. Statistics were calculated from unpaired t-test with ** indicated p<0.01. (E) Immunofluorescence images of endogenous TMED9 in control siRNA or siTMED9-treated YFP-PrP* NRK cells that were depleted for TMED9. For each condition, cells were treated with thapsigargin (TG) for 0 min or 90 min before fixation and staining for TMED9 and ER-resident chaperone, calnexin (CNX). Nuclei were stained with Hoechst. Larger fields of views for these panels and additional data for these experiments are presented in . Scale bar represents 10 µm. (F) Plot of the average Pearson’s r values between YFP-PrP* and endogenous calnexin (CNX) for control (CTL) siRNA or siTMED-treated cells at 0 min or 90 min after thapsigargin addition, as shown in (E). The number of cells analyzed for each condition are listed as follows: CTL siRNA 0 min post-TG-treatment (n=14), CTL siRNA 90 min post-TG-treatment (n=12), siTMED9 0 min post-TG-treatment (n=15), siTMED9 90 min post-TG-treatment (n=14). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CNX, within the boundaries of the cell as defined by the outline of the CNX staining. (G) Immunofluorescence images of endogenous TMED9 in control (CTL) or TMED9 siRNA (siTMED9)-treated YFP-PrP WT NRK cells that were depleted for TMED9. Cells were fixed and stained for TMED9 and ER-resident chaperone, calnexin (CNX) at steady-state conditions. Nuclei were stained with Hoechst. Scale bar represents 10 µm. (H) Plot of the average Pearson’s r values between YFP-PrP WT and endogenous calnexin (CNX) for control (CTL) siRNA or siTMED-treated cells at steady-state, as shown in (G). The number of cells analyzed for each condition are listed as follows: CTL siRNA (n=22) and siTMED9 (n=24). Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and CNX, within the boundaries of the cell as defined by the outline of the CNX staining.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control, Standard Deviation, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Immunofluorescence image stained for endogenous TMED9 and CNX in control YFP-PrP* NRK (CTL) or siTMED9-treated YFP-PrP* NRK cells (siTMED9 cells) at steady state (TG 0min) after 90 min of TG-treatment (TG 90 min). Nuclei were stained with Hoechst. For the siTMED9-treated cells, large white arrows point to cells with only partial TMED9 knockdown. Scale bar represents 10 µm. Scale bar represents 10 µm. (B) Representative western blots of either control (CTL) or TMED9 siRNA (siTMED9)-treated GFP-CD59 (C94S) NRK cells that were either untreated (TG –) or treated with 90 min TG (TG +) (n=3 biological replicates). (C) Bar graph representing the mean band intensity for GFP-CD59 (C94S) from 3 biological replicates as shown in (B). For each condition, GFP-CD59 (C94S) band intensities were double normalized. First, GFP band intensity was normalized against the band intensity of GAPDH. Second, the GFP band intensity was normalized against the untreated control (“siTMED9 – TG – “) band intensity. Error bars represent standard deviation of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** indicated p<0.01. (D) Time-lapse images of control (CTL) or TMED9 siRNA (siTMED9) GFP-CD59 (C94S) NRK cells. Image-collection was started immediately after the addition of thapsigargin (TG). Scale bar represents 20 µm. (E) Percentage of cells showing a perinuclear-pattern for GFP-CD59 (C94S), indicative of Golgi-localization, after 30 min of TG-treatment. These data are derived from 4 independent experiments (n=4). Symbols were coded for each independently performed experiment. The number of cells analyzed for each biological replicate are as follows (CTL: triangle 37, diamond 59, circle 18, square 54; siTMED9: triangle 73, diamond 93, circle 28, square 64). Error bars represent standard deviation of the mean. Statistics were calculated from unpaired t-test with Welch’s correction with ** indicated p<0.01.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Knockdown, Western Blot, Standard Deviation, Derivative Assay

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of either non-targeting siRNA (CTL) or TMED9 siRNA (siTMED9)-treated YFP-PrP* NRK cells. Membranes were probed for TMP21 and GAPDH or TMED2 and GAPDH. (n=3 biological replicates). (B) Bar graph representing the mean band intensity for TMED9, TMP21 or TMED2 from 3 independently performed experiments as shown in (A). For each condition, TMED9. TMP21 or TMED2 band intensities were double normalized: first against the band intensity of GAPDH, second against the CTL band intensity. Error bars represent the standard deviation of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05. (C) Time-lapse images of YFP-PrP* NRK cells that were co-transfected with mCh-Sec61β. Cells were either not pretreated (Control) or pretreated with BRD4780 (BRD pretreat) for 30 min. Image collection was started immediately after the addition of thapsigargin (TG). Scale bars represent 10 µm. (D) Plot of the average Pearson’s r values between YFP-PrP* and mCh-Sec61β. For Control vs. BRD4780-pretreated conditions, as described in (C), 10 time-lapses of individual cells were analyzed for the 0 and 30 min time points. Pearson’s colocalization coefficients, r, were measured between YFP-PrP* and mCh-Sec61β within the boundaries of the cell. For each data point, the boundaries of each cell at 0 and 30min time points were revealed by temporarily maximizing the gain for YFP-PrP*. (E) Representative western blots of YFP-PrP* NRK cells that were untreated (CTL), TG-treated for 90 min (TG) or BRD4780 (30 min)-pretreated and harvested after 90 min of TG treatment (BRD+TG) (n=3 biological replicates). Blots were probed for TMED9, PrP (YFP-PrP*) and GAPDH. (F) Average of normalized YFP-PrP* band intensity from 3 independently performed experiments as shown in (E). For each condition, YFP-PrP* band intensities were double normalized. First, they were normalized against the band intensity of GAPDH. Second, they were normalized against the CTL band intensity. Error bars represent standard deviations of the mean. Statistics were calculated from unpaired t-test with Welch’s correction with *** indicated p<0.001. (G) Representative western blots of YFP-PrP* NRK cells that were either untreated control (CTL) or treated with BRD4780 for 30 min (30’ BRD). Blots were probed for TMED9, TMP21, and TMED2 and their respective GAPDH (n=3 biological replicates). TMED9 protein profile shows 2 distinct bands, one lower band (orange arrow, LMW) and a higher band (black arrow, HMW). (H) Average of normalized High Molecular Weight (HMW) and Low Molecular Weight (LMW) TMED9 band intensity from 3 independently performed experiments as shown in (G). For each condition, TMED9 band intensities were double normalized. First, they were normalized against the band intensity of GAPDH. Second, they were normalized against the CTL intensity of each protein probed. Orange bars represent the quantification of the lower molecular weight (LMW) band intensity and gray bars represent the higher molecular weight (HMW) band intensity. Error bars represent standard deviations of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05, and *** p<0.001. (I) Representative western blots of endogenous TMED9 from YFP-PrP* NRK cells after 180 min of BRD-treatment (n=3 biological replicates). Blots were probed for TMED9 and GAPDH. TMED9 protein profile shows 2 distinct bands, one LMW (orange arrow) and a HMW (black arrow). (J) Average of normalized HMW and LMW TMED9 band intensity from 3 independently performed experiments as shown in (I). For each time point TMED9 band intensities were double normalized: first by the band intensity of GAPDH and second against the BRD4780 0 min band intensity. Orange bars representing the measurement for the LMW of TMED9 and gray, the HMW. Error bars represent standard deviations of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** p<0.01. (K) Representative western blots of TMP21, TMED2 and their representatives GAPDH either control (CTL) or BRD4780 (30 min)-treated YFP-PrP* NRK cells (n=3 biological replicates). (L) Average of normalized TMP21 and TMED2 band intensities from 3 independently performed experiments as shown in (K). For each condition, TMP21 or TMED2 band intensities were double normalized: first against the band intensity of GAPDH, second against the CTL band intensity. Error bar represents standard deviation of the mean. Symbols were coded for each independently performed experiment. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Standard Deviation, Transfection, Control, High Molecular Weight, Molecular Weight

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Representative western blots of YFP-PrP* NRK cells that were treated as listed below (n=3 biological replicates). Untreated (Control), treated with BRD4780 for 180 min (BRD), pretreated with bafilomycin A1 (BAF) for 180 min and co-treated with BRD for 180 min (BAF+BRD), pretreated with MG132 for 180 min and co-treated with BRD for 180 min (MG132+BRD), pretreated with 3-methyladenine (3MA) for 180 min and co-treated with BRD for 180 min (3MA+BRD) or pretreated with BFA for 180 min and co-treated with BRD for 180 min (BFA+BRD). Blots are probed for endogenous TMED9 and GAPDH. (B) Bar graph representing the mean band intensity for TMED9 from 3 biological replicates as shown in (A). For each condition, TMED9 band intensities were double normalized first by the band intensity of GAPDH and second within individual experiments using control band intensity. Orange bars represent the measurement for the low molecular weight (LMW) band of TMED9 and gray bars represent the measurement of the high molecular weight (HMW) band of TMED9. Error bars represent standard deviations of the mean. Symbols were coded for individual experiments. Statistics were calculated from unpaired t-test with Welch’s correction with * indicated p<0.05 and ** p<0.01. (C) Western blots of digested protein lysates of stably transfected YFP-PrP* NRK cells either in control condition (Control) or after a pretreatment of 2 h of bafilomycin followed by co-incubation with 2 h of BRD4780 (BAF+BRD). Protein lysates undigested (water) or digested with Endoglycosidase H (Endo H), Neuraminidase+O-Glycosidase (NA+O-Glyc.), Peptide-N-glycosidase F (PNG) or NA+O-Glyc+PNGase (NA+O-G+PNG) for 3 h at 37°C (n=1 experiment). Blots were probed for TMED9 and calnexin (CNX). (D) Western blot band graphics obtained by first analyzing the plot lanes and second generating the line graphs of pre-selected regions of interest (ROI) from the western blot for control samples (left) or BAF+BRD samples (right). Black arrow represents the HMW form, orange arrow represents the LMW and purple, the unglycosylated form of TMED9. (E) (left) Fluorescence image of a typical untreated YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and ER-marker, mCherry-Sec61β. Scale bar represents 10 µm. (right) Magnified panel of the stroked region on the right. Scale bar represents 1 µm. (F) (left) Fluorescence image of a typical untreated YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and Golgi marker, FusionRed-SiT. Scale bar represents 10 µm. (right) Magnified panel of the stroked region on the right. Scale bar represents 1 µm. (G) Time-lapse imaging sequence of a NRK cell that was co-transfected with CER-TMED9 and ER marker, mCherry-Sec61β. Image-collection was started immediately after the addition of 100 µM BRD4780 treatment. Scale bar is 10 µm. (H) Example of an ER mask created from the ER-marker, mCherry-Sec61β. The ER mask was used to measure the CER-TMED9 fluorescence intensity within the ER at 0 or 30 min time points after the addition of BRD4780 from 9 individual time-lapses as shown in (G) and quantified in (I). (I) Bar graph representing the mean of ER-localized CER-TMED9 fluorescence-intensity measurements of 9 individual time-lapses of CER-TMED9 and mCherry-Sec61β-expressing cells, as shown in (G). The ER mask was generated based on and as analyzed in in (H). Statistics were calculated from unpaired t-test with Welch’s correction with ** indicated p<0.01 (J) Representative immunofluorescence images of YFP-PrP* NRK cells that were transfected with CER-TMED9 under control untreated (CTL) or 100 µM BRD4780 (30min)-treated (BRD) conditions and stained for GM130. Scale bar is 20 µm. (K) Bar graph representing the mean of Pearson’s correlation coefficient r to measure colocalization between CER-TMED9 and GM130 under untreated (CTL) and BRD4780 (30 min)-treated (BRD) conditions, as exemplified in (J). Colocalization for 18 cells were measured for control conditions and 11 cells were measured for BRD4780 (30min)-treated cells. Statistics were calculated from unpaired t-test Welch’s correction with *** indicating p<0.001.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control, Molecular Weight, High Molecular Weight, Stable Transfection, Transfection, Incubation, Fluorescence, Marker, Imaging, Sequencing, Expressing, Generated, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Western blots of digested lysates of YFP-PrP* NRK cells in control and BRD4780-treated conditions. Protein lysates were not digested (water) or digested with Endoglycosidase H (Endo H), Neuraminidase only (NA), Neuraminidase+O-Glycosidase (NA+O-G.), Peptide-N-glycosidase F (PNG) or Neuraminidase+O-Glycosidase + Peptide-N-glycosidase F (NA+O-G+PNG) for 3 h at 37°C (n=1 experiment). Blots were probed for TMED9 and calnexin (CNX). (B) Western blot band graphics were obtained by first analyzing the plot lanes and second by generating line graphs of pre-selected regions of interest (ROI) from the western blot for control samples (left) or BRD4780 (30 min)-treated samples (right). Black arrow represents the HMW form, orange arrow represents the LMW and purple, the unglycosylated form of TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Table presenting the different treatments and their effects on TMED9’s molecular weight. As depicted in the treatment time-course, YFP-PrP* NRK cells were pre-treated with BRD4780 (BRD) or cycloheximide + BRD4780 (CHX BRD) for different periods of time with or without a 30 min BRD4780 wash-out (wash). “CHX wash” indicates that after pre-treatment with CHX + BRD, the BRD4780 wash-out included cycloheximide. The numbers below the treatment time-course diagram indicate the percentage of cells that were competent for RESET after each treatment course. The percentages were obtained by dividing the number of cells in which YFP-PrP* relocalized from the ER to the Golgi within 30 min of adding thapsigargin by the total number of cells counted. The n values (i.e. total number of cells counted per treatment course) were included directly under the percentage. We assessed “ER” vs “Golgi” localization based on the pattern of YFP fluorescence. At steady-state YFP-PrP* fluorescence is dispersed throughout the cell in a reticulo-tubular pattern with a sharp outline of nucleus that is consistent with ER and the nuclear envelope subdomain of the ER. During RESET, YFP-PrP* fluorescence changes from disperse a perinuclear localization that is consistent with the Golgi. Aligned directly below the treatment courses and percentages, are the TMED9 and GAPDH western blots. Each lane encompassing the treatment, % RESET competent cells, TMED9 molecular weight, GAPDH loading control, was numbered on the bottom. There were 10 individual treatment conditions. (B) Representative images of YFP-PrP* NRK cells after each indicated treatment conditions (1-10). The outline of each cell has been traced in yellow using the ROI tool in Image J.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Molecular Weight, Fluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: (A) Still frames of a typical YFP-PrP* NRK cell that was co-transfected with CER-TMED9 and mCherry-Sec61β and treated as follows. The first image is of the cell before treatment began (Untreated). The second image is of the cell after 30 min with 50 µg/ml of CHX + BRD4780 (CHX + BRD4780 30 min). The third image is of the cell 30 min after washing with CHX-only medium (CHX wash 30 min). The fourth image is of the cell 30 min after treatment with 50 µg/ml of CHX + 0.1 µM thapsigargin (CHX + TG 30 min). Juxtaposed with each image is an area zoom of the region within the stroked frame. Scale bar represents 10 µm in the larger field of view and in the magnified panel of the stroked region. These select images are also provided in Video 2. (B) Representative western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or YFP-PrP* NRK cells (n=3 biological replicates). Quantified in (C). Cells were harvested at the indicated time points after BRD4780-treatment. Blots were probed for PrP to detect YFP-PrP*, or for endogenous calnexin (CNX) and TMED9. (C) Bar graph representing the mean band intensity for CNX and TMED9 in the eluates from 3 independently performed experiments as shown in (B). For each time point, CNX and TMED9 eluate band intensities were double normalized. First, they were normalized by the band intensity of the eluted YFP-PrP* band. Second, they were normalized by the time 0 eluate band intensity for each protein probed. Error bars represent standard deviations of the mean. Symbols were coded for individual experiments. Statistics were calculated from unpaired t-test with Welch’s correction with and ** indicated p<0.01. (D) Western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or YFP-PrP* NRK cells (n=1 experiment). Cells were harvested after the indicated treatments. Blots were probed for PrP to detect YFP-PrP*, or for endogenous calnexin (CNX) and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: Time-lapse images of a typical YFP-PrP* NRK cell that was transfected with CER-TMED9. Image-collection was started immediately after the addition of 100 µM BRD4780 treatment. Scale bar represents 20 µm.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Transfection

Journal: bioRxiv

Article Title: The p24-family member, TMED9, clears misfolded GPI-anchored proteins from the ER to the Golgi via the Rapid ER Stress-Induced Export pathway

doi: 10.1101/2024.09.27.615420

Figure Lengend Snippet: Western blots of GFP-tag co-immunoprecipitations (co-IPs) from the parental untransfected NRK cells (P) or stably transfected YFP-PrP* NRK cells (n=1 experiment). YFP-PrP* NRK cells were treated with 100 µM BRD4780 (BRD) and collected for co-IP at the indicated time points. Cells were harvested at the indicated time points for co-immunoprecipitation of YFP-PrP* with anti-GFP antibody conjugated beads in addition to GFP to detect co-immunoprecipitation of YFP-PrP* constructs, blots were probed for endogenous calnexin (CNX), TMED2, TMP21, and TMED9.

Article Snippet: The following primary antibodies were added to the TBS-T + 2.5% milk solution at the following concentrations: mouse monoclonal αGFP (Proteintech, 66002-1-IG) at 1:2000, rabbit polyclonal αTMED9 (Proteintech, 21620-1-AP) at 1:2500, rabbit polyclonal αTMP21 (homemade and described previously [ ]) at 1:2000, rabbit polyclonal αTMED2 (Proteintech, 11981-1-AP) at 1:2000, mouse monoclonal αCalnexin (Proteintech, 66903-1) at 1:2500, rabbit polyclonal αPrP (Proteintech, 12555-1-AP) at 1:2500, mouse monoclonal αGAPDH antibody (ThermoFisher, MA5-15738) at 1:4000, for 2 hours at room temperature or overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Construct