Journal: Life Science Alliance

Article Title: Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides

doi: 10.26508/lsa.202101060

Figure Lengend Snippet: (A) Construct map for tagged type I pre-procollagen. The tagged pre-procollagen protein, Gr-CC, includes GFP in the repeating structure and mCherry in C-pp. The other constructs, including GFP in the repeating structure (designated as Gr), and GFP in N-pp and mCherry in C-pp (designated as GN-CC), were also used in this study. White triangles indicate the cleavage points. (B) Confocal microscopy of the tagged procollagen, Gr-CC, in a cell. Of note, the perinuclear region has procollagen in yellow and processed collagen in green indicated by arrows. Scale bars: 10 μm. (C) Immunoelectron microscopy of cells expressing Gr-CC. Samples were reacted with an anti-GFP antibody. The lower left is a negative control, which was reacted without the primary antibody. ER, endoplasmic reticulum; Ly, lysosome; Mi, mitochondria; Nu, nuclear. (D) Confocal microscopy of NIH3T3 cell expressing Gr-CC with the collagen trafficking assay. The cell was incubated at 40°C for 3 h, and immunostained with an anti-calreticulin antibody. The region surrounded by the yellow square in the lower panel was magnified. Scale bars: 10 μm (B, D). (E) Confocal microscopy of a NIH3T3 cell expressing Gr-CC, which was incubated at 20°C or 37°C and immunostained with anti-TGN46 antibody. Note that almost all of GFP signals were colocalized with TGN46 at 20°C in the presence of cycloheximide. Scale bars: 5 μm. (F) Detection of tagged procollagen by a Western blot analysis. NIH3T3 cells transfected with Gr-CC (lane 2) and GN-CC (lane 3) were lysed with NativePAGE sample buffer and subjected to analysis. Upper panel: Immunoblotted with an anti-human collagen I antibody; lower panel: Immunoblotted with the anti-GFP antibody. An identical membrane was used for reproving by another antibody to confirm identical signal positions. Arrows indicate tagged procollagens; arrowheads indicate endogenous collagen. (G) Detection of tagged procollagen by a blue native-PAGE analysis. Samples from (F) were subjected to the analysis and reacted with the anti-GFP antibody.

Article Snippet: Antibodies used for immunoblotting were as follows: anti-collagen type I antibodies from Rockland and Boster Biological Technology; an anti-α-tubulin antibody (T9026) from Sigma-Aldrich; an anti-GFP antibody from Thermo Fisher Scientific; and an anti-mCherry antibody from Novus Biological.

Techniques: Construct, Confocal Microscopy, Immuno-Electron Microscopy, Expressing, Negative Control, Incubation, Western Blot, Transfection, Membrane, Blue Native PAGE

Journal: Life Science Alliance

Article Title: Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides

doi: 10.26508/lsa.202101060

Figure Lengend Snippet: (A) Confocal fluorescence microscopy of pseudopodia of a cell expressing the tagged collagen. Human primary fibroblast cells transfected with Gr-CC were seeded on a fibronectin-coated dish and cultured for 24 h. Magnification: ×40. Scale bar: 1 μm. Of note, the red channel to detect mCherry had no signal. (B) Magnification of fibrils including tagged type I collagen between cells. NIH3T3 cells transfected with Gr regulated by the CMV promoter were cultured for 1 mo and fibrils between cells were detected by transmission electron microscopy. The upper panel shows horizontal sections and the lower shows cross-sections. The red number indicates the diameter of each fibril. Magnification: ×20,000 and ×40,000, respectively. (C) Detection of GFP-tagged collagen secreted from NIH3T3 cells in collagen fibrils by an immunoelectron microscopic analysis. The left panel shows fibrils among cells stably expressing Gr-CC detected by an anti-GFP antibody followed by a 15-nm gold-anti-rabbit antibody. The right panel shows the negative control without the primary antibody. Scale bars: 200 nm. Note that colloidal gold signals were specifically detected in the left panel. (D) Detection of collagen fibrils secreted from NIH3T3 cells expressing Gr as in (B) by atomic force microscopy. Left: original image. Right: contrast was adjusted. The axial repetitive structure in the fibril reflects the collagen-specific structure (C, D).

Article Snippet: Antibodies used for immunoblotting were as follows: anti-collagen type I antibodies from Rockland and Boster Biological Technology; an anti-α-tubulin antibody (T9026) from Sigma-Aldrich; an anti-GFP antibody from Thermo Fisher Scientific; and an anti-mCherry antibody from Novus Biological.

Techniques: Fluorescence, Microscopy, Expressing, Transfection, Cell Culture, Transmission Assay, Electron Microscopy, Stable Transfection, Negative Control

Journal: Life Science Alliance

Article Title: Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides

doi: 10.26508/lsa.202101060

Figure Lengend Snippet: (A) Snapshots indicating the intracellular distribution of C-pp (left) and the repeating structure domain (middle). The two results were merged in the right panel. ×20 magnification. The original live imaging of C-pp is shown in . The cell on the right transferred the repeating structure domain by pseudopodium, whereas C-pp accumulated in the perinuclear region. (B) Western blot analysis of C-pp. Lysates prepared from cells expressing Gr-CC or GN-CC were separated by SDS–PAGE and reacted with an anti-mCherry (upper panel) or anti-tubulin (lower panel) antibody, respectively. *: specific signals for C-pp+mCherry. The right panel shows the control lysate of cells expressing mCherry. (C) Detection of secreted proteins, including mCherry, in the culture medium. Cell culture media used for cells expressing mock, Gr-CC, and GN-CC were separated by SDS–PAGE and reacted with the anti-mCherry antibody. The right panel shows control media without cells, and the lysates of cells expressing GFP and mCherry. (D) Intracellular distribution of N-pp and C-pp. Snapshots from the live imaging of NIH3T3 cells expressing GN-CC. The original live imaging of the merged panel is shown in . The lower left panel shows the upper section of cells expressing GN-CC visualized on confocal microscopy. Note that the cell top is red. The lower right panel shows an enlarged part of the lower middle panel. Scale bars: 20 μm in (A) and (D). (E) Western blot analysis of N-pp. The same experiments were performed as in (B) with an anti-GFP antibody. In the present study, Gr-CC lacking the processing site for C-pp (designated as Gr-CC*) was used to detect the fragment consisting of the repeating structure domain+C-pp. *: a specific signal in GN-CC. **: a signal relative to the fragment consisting of the repeating structure domain+C-pp. Arrows indicate the fragments consisting of the N-pp+repeating structure domain and the repeating structure domain only. (F) Detection of the secreted protein including GFP in the culture medium. The same culture media as in (C) were reprobed with the anti-GFP antibody. The arrow indicates the processed repeating structure domain with the GFP tag in the Gr-CC lane. (G) Comparison of the collagen/procollagen ratio between the lysate and culture medium of cells expressing Gr-CC. The signal intensities of Western blot analyses with the anti-GFP antibody were measured. Each value represents the mean ± SD of triplicate measurements. * P < 0.01. All experiments were performed using NIH3T3 cells (A, B, C, D, E, F, G).

Article Snippet: Antibodies used for immunoblotting were as follows: anti-collagen type I antibodies from Rockland and Boster Biological Technology; an anti-α-tubulin antibody (T9026) from Sigma-Aldrich; an anti-GFP antibody from Thermo Fisher Scientific; and an anti-mCherry antibody from Novus Biological.

Techniques: Imaging, Western Blot, Expressing, SDS Page, Control, Cell Culture, Confocal Microscopy, Comparison

Journal: Life Science Alliance

Article Title: Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides

doi: 10.26508/lsa.202101060

Figure Lengend Snippet: Related to . Lysosomes were stained with LysoBrite. The region surrounded by the red square in the lower right panel was magnified. Dashed circles indicate lysosomes containing many C-pp, but only a few repeating structure domains. Plot profiles of the colocalization of mCherry and GFP with lysosomes show 21.1% and 5.94% of each signal in the cell, respectively. Scale bars: 20 μm.

Article Snippet: Antibodies used for immunoblotting were as follows: anti-collagen type I antibodies from Rockland and Boster Biological Technology; an anti-α-tubulin antibody (T9026) from Sigma-Aldrich; an anti-GFP antibody from Thermo Fisher Scientific; and an anti-mCherry antibody from Novus Biological.

Techniques: Staining

Journal: Life Science Alliance

Article Title: Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides

doi: 10.26508/lsa.202101060

Figure Lengend Snippet: Related to and and and . Lysates prepared from CFSC-5H cells expressing mock, Gr-CC, or GN-CC were separated by SDS–PAGE and detected with an anti-mCherry (upper panel) or anti-tubulin (left, lower panel) antibody, respectively. The molecular weight of the processed C-pp+mCherry fragment is at ∼60 kD (see ). The right panel confirms no new band less than 60 kD in Gr-CC and GN-CC lanes with prolonged exposure times. *: signals for procollagen tagged with GFP and mCherry.

Article Snippet: Antibodies used for immunoblotting were as follows: anti-collagen type I antibodies from Rockland and Boster Biological Technology; an anti-α-tubulin antibody (T9026) from Sigma-Aldrich; an anti-GFP antibody from Thermo Fisher Scientific; and an anti-mCherry antibody from Novus Biological.

Techniques: Expressing, SDS Page, Molecular Weight

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of two N-terminal regions of the OsPSY2 protein including the hydrophobic helix (HH) 1 on subcellular localization in rice protoplasts. (A) Individual and merged images of sGFP, chlorophyll, and visible protoplasts using confocal microscopy. sG and RTp-sG were used as a cytosolic (Cy) and stromal (St) localization markers, respectively. Scale bars indicate 5 μm. (B) Western blot analysis to compare the amount and size of sGFP. Rice protoplasts were cotransfected with each experimental vector and an OsPSY2:mC vector as a transfection control. This blot was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (pink rectangle), and anti-rice actin (marked at 45 kDa, to be used as an internal reference of the total protein amount). (C) Prediction of cleavage probability in the N-terminal 80-amino-acid region of OsPSY2 as implemented by the TargetP 2.0 program ( https://services.healthtech.dtu.dk/service.php?TargetP ). Estimated cleavage scores of the 80 amino acids of PTp are shown in Supplementary Table S1 .

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of the hydrophobic region (HR) 2 on subcellular and suborganellar localization in rice protoplasts. (A) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. PTp-sG, OsPSY2:mC, and mC were used as markers for dual localization into plastoglobules (PGs) and stroma (St) as well as single localization into a PG and the cytosol (Cy), respectively. Scale bars indicate 5 μm. (B) Western blot analysis to confirm the cleavage in HR2 when placed right after a Tp signal such as RTp or HR1 within PTp. (C) Western blot analysis to confirm that no cleavage occurred in HR2 when placed at the C-terminus of sGFP. In both (B, C) , rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector used as a transfection control. In one exceptional case we used RTp-sG-HR2-mC with intrinsic mC. The blot shown in (B) was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (red diamond for the experimental vector or pink rectangle for the control vector), and anti-rice actin (marked at 45 kDa and used as an internal reference indicating total protein amount). The blot shown in (C) was performed with two antibodies simultaneously: Anti-GFP and anti-rice actin in the left panel, and anti-mCherry and anti-rice actin in the right panel, respectively. The expected protein size is noted under the blot.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Plasmid Preparation, Transfection, Control

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Effect of the hydrophobic region (HR) 3 on PG localization in rice protoplasts. (A) Individual and merged images of mCherry, chlorophyll, and visible protoplasts using confocal microscopy. OsPSY2:mC and OsPSY2(ΔPTp):mC were used as localization markers of plastoglobules (PGs) and the cytosol (Cy), respectively. Scale bars indicate 5 μm. (B) Western blot analysis to confirm the presence of a PG-targeting signal in an area other than PTp in OsPSY2. Rice protoplasts were individually transfected with three experimental vectors. This blot was conducted with anti-mCherry (red diamond) and a polyacrylamide gel electrophoresis (PAGE) image is shown to indicate total protein amounts. (C) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. RTp-sG and OsPSY2:mC were used as localization markers for the stroma (St) and plastoglobules (PGs), respectively. Scale bars indicate 5 μm. (D) Western blot analysis to reveal the precise location of HR3 that exhibits PG-targeting capacity. Rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector, used as a transfection control. The blot was performed with three antibodies simultaneously: Anti-GFP (blue circle), anti-mCherry (pink rectangle), and anti-rice actin (marked at 45 kDa and used as an internal reference of total protein amount). (E) Quantitative real-time-PCR to indicate relative transcript levels depending on different protein levels for the three vectors. The rice ubiquitin gene (AK061988) was used as a reference to normalize RNA amounts. qRT-PCR was performed using three technical replicates with three biological samples per vector.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Western Blot, Transfection, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Quantitative RT-PCR

Journal: Frontiers in Plant Science

Article Title: Stepwise protein targeting into plastoglobules are facilitated by three hydrophobic regions of rice phytoene synthase 2

doi: 10.3389/fpls.2023.1181311

Figure Lengend Snippet: Differential effects of the hydrophobic region (HR) 3 on the PG-localization depending on PTp in rice protoplasts. (A) Individual and merged images of sGFP, mCherry, chlorophyll, and visible protoplasts using confocal microscopy. OsPSY2:mC was used as a plastoglobule (PG) localization marker. Scale bars indicate 5 μm. (B) Western blot analysis to determine the factors influencing aggregation and loss of fluorescence. Rice protoplasts were cotransfected with an experimental vector and an OsPSY2:mC vector used as a transfection control. In one exceptional case PTp-sG-HR3-mC was used, which has intrinsic mC. The blot was performed with two antibodies simultaneously: Anti-GFP (blue circle) and anti-rice actin (marked at 45 kDa) on the left panel, and anti-mCherry (red diamond for experimental vector or pink rectangle for the control vector) and anti-rice actin on the right panel, respectively.

Article Snippet: Next, a Western blot was performed using an anti-rabbit polyclonal antibody against GFP (Abcam, Cambridge, UK), an antibody against mCherry (Novus Biologicals, Denver, USA), and an antibody against actin (Abcam, Cambridge, UK).

Techniques: Confocal Microscopy, Marker, Western Blot, Fluorescence, Plasmid Preparation, Transfection, Control