rabbit polyclonal lc3 pm036 antibody  (MBL International)

Journal: International Journal of Molecular Sciences

Article Title: Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3

doi: 10.3390/ijms251910707

Figure Lengend Snippet: Autophagy activation to scavenge protein aggregates in ATXN3-Q 75 -GFP SH-SY5Y cells. Cells were treated with trehalose (10 mM) or studied compounds (10 μM), as described in . On day 8, LC3 puncta, ATXN3-Q 75 aggregates, and LC3-II:LC3-I ratio were assessed. ( a ) Confocal microscopy images of LC3-positive puncta and ATXN3-Q 75 aggregates in ATXN3-Q 75 -GFP SH-SY5Y cells treated with trehalose or studied compounds (green, expressed ATXN3-Q 75 -GFP protein; red, LC3; blue, DAPI-stained nuclei). The arrows (white) in the uninduced photo indicate puncta. The arrows (orange) in the trehalose-treated photo indicate overlapped puncta and aggregates. Shown below are analyses of LC3 puncta and ATXN3 aggregates per cell, and colocalization coefficient of ATXN3-Q 75 -GFP with LC3 puncta. ( b ) Immunofluorescence staining of LC3 and P62 in ATXN3-Q 75 -GFP SH-SY5Y cells treated with trehalose or studied compounds (red, LC3; yellow, P62; blue, DAPI-stained nuclei). The arrows (orange) in the trehalose-treated photo indicate overlapped puncta and P62. Shown below is colocalization coefficient of LC3 with P62. ( c ) Relative LC3-I and LC3-II levels analyzed by Western blot using LC3 and GAPDH (as a loading control) antibodies. Relative LC3-II:LC3-I ratio in uninduced cells was set at 100% ( n = 3). p values: with vs. without doxycycline induction (###: p < 0.001), or with vs. without compound treatment (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Article Snippet: The input and eluted proteins were separated on a 10% SDS–polyacrylamide gel and immunoblotted with LC3 (1:2000; MBL International #PM036, Woburn, MA, USA), GFP (1:1000; Bioman #egfp001r, New Taipei City, Taiwan), ATXN3 (1:1000; Invitrogen #PA5-26251), or expanded polyQ (5TF1-1C2, 1:1000; Merck #MAB1574) antibody at 4 °C overnight.

Techniques: Activation Assay, Confocal Microscopy, Staining, Immunofluorescence, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3

doi: 10.3390/ijms251910707

Figure Lengend Snippet: Interaction of studied compounds with ATXN3-Q 75 and LC3. ( a ) pcDNA3-TXN3-Q 14−75 -VC and pcDNA5-VN-LC3 constructs and amino acid sequences of the expressed recombinant proteins. To produce ATXN3-Q 14−75 -VC, a 105-amino acid ATXN3-Q 14 or a 166-amino acid ATXN3-Q 75 -containing Eco RI- Nco I DNA fragments (marked in red: ATXN3-Q 75 ) was linked to a 28-amino acid VC (C-terminal fragment of Venus)-containing Nco I- Not I DNA fragment (marked in green) through a 6-amino acid linker (containing Nco I restriction site). To produce VN-LC3, a 211-amino acid VN (N terminal fragment of Venus)-containing Bam HI- Xba I DNA fragment (marked in green) was linked to a 125-amino acid LC3-containing Xba I- Bam HI DNA fragment (marked in orange) through a 2-amino acid linker (containing Xba I restriction site). ( b ) Co-immunoprecipitation (Co-IP) of ATXN3-Q 14−75 -VC and VN-LC3: 293T cell lysates containing 100 μg of ATXN3-Q 14 -VC, ATXN3-Q 75 -VC, or VN-LC3, and mixtures of ATXN3-Q 14−75 -VC and VN-LC3 were subjected to co-IP. The expression of ATXN3-Q 14−75 -VC or VN-LC3 fusion proteins in cell lysates were confirmed by anti-ATXN3 (detecting ATXN3-Q 14−75 -VC protein), anti-polyQ (5TF1-1C2, detecting only ATXN3-Q 75 -VC protein), or anti-LC3 and anti-GFP (detecting VN-LC3 protein) antibody staining of protein blot. ( c ) The 100 μg individual (ATXN3-Q 75 -VC or VN-LC3) or combined cell lysates in the presence or absence of the test compound (100 μM) were immunoprecipitated with 5TF1-1C2 antibody, and the presence of VN-LC3 protein was detected by immunoblot analysis with anti-GFP antibody. Mouse IgG antibody was used as a negative control in the co-IP experiment. The experiments were repeated three times with similar results.

Article Snippet: The input and eluted proteins were separated on a 10% SDS–polyacrylamide gel and immunoblotted with LC3 (1:2000; MBL International #PM036, Woburn, MA, USA), GFP (1:1000; Bioman #egfp001r, New Taipei City, Taiwan), ATXN3 (1:1000; Invitrogen #PA5-26251), or expanded polyQ (5TF1-1C2, 1:1000; Merck #MAB1574) antibody at 4 °C overnight.

Techniques: Construct, Recombinant, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Staining, Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3

doi: 10.3390/ijms251910707

Figure Lengend Snippet: Autophagic degradation of expanded polyQ protein in 293T cells with dual expression of VN-LC3 and ATXN3-Q 75 -VC proteins. ( a ) pTRE3G-BI-VN-LC3-ATXN3-Q 14−75 -VC construct for doxycycline-inducible dual expression of VN-LC3 and ATXN3-Q 14−75 -VC. The pTRE3G-BI promoter consists of 7 repeats of a 19 bp tetracycline (tet) operator sequence. VN-LC3 was cloned in Bam HI site of multiple cloning site (MCS)-1; ATXN3-Q 14−75 -VC was cloned between Xba I and Kpn I sites of MCS-2. ( b ) Experimental flow chart. On day 1, 293T cells were plated, and they were co-transfected with pTRE3G-BI-VN-LC3-ATXN3-Q 14−75 -VC and pCMV-Tet3G (4:1 ratio) on day 2. Doxycycline (1 μg/mL) alone or along with test compound (10 μM) was added to cells to induce trans-activator protein and hence ATXN3-Q 14−75 -VC and VN-LC3 expression on day 3. At 24 h post-induction, cell lysates were prepared for immunoblot analysis of VN-LC3 and ATXN3-Q 14−75 -VC protein expression. In addition, high-content analysis of overlapped 1C2 (ATXN3-Q 75 )/Venus signal was performed at 6, 9, 12, and 24 h post-induction. ( c ) Immunoblot analysis of ATXN3-Q 14−75 -VC (anti-ATXN3 antibody) and VN-LC3 (anti-LC3 antibody) at 24 h post-induction. The red asterisk indicates antibody-stained ATXN3-Q 75 -VC, VN-LC3, or ATXN3-Q 14 -VC proteins. ( d – f ) High-content images (Venus–BiFC, green; ATXN3-Q 75 -1C2 stain, red) of 293 transfected cells at 24 h ( d , e ) or 6, 9, 12, and 24 h post-induction of protein expression (f). Nuclei were counterstained with DAPI (blue). The arrows in ( e ) indicate split-Venus (white arrow), 1C2-stained ATXN3-Q 75 aggregates (white arrow), and overlapped split-Venus and ATXN3-Q 75 aggregates (orange arrow). ( g ) High-content images and analyses of IC2 and Venus (left) and Venus-tagged LC3 puncta (right) in compound-treated transfected cells at 24 h post-induction of protein expression. Nuclei were counterstained with DAPI (blue). The arrows (orange) in the induced photo indicate overlapped IC2 and split-Venus. p values: with vs. without doxycycline induction (###: p < 0.001), or with vs. without compound treatment (*: p < 0.05).

Article Snippet: The input and eluted proteins were separated on a 10% SDS–polyacrylamide gel and immunoblotted with LC3 (1:2000; MBL International #PM036, Woburn, MA, USA), GFP (1:1000; Bioman #egfp001r, New Taipei City, Taiwan), ATXN3 (1:1000; Invitrogen #PA5-26251), or expanded polyQ (5TF1-1C2, 1:1000; Merck #MAB1574) antibody at 4 °C overnight.

Techniques: Expressing, Construct, Sequencing, Clone Assay, Cloning, Transfection, Western Blot, High Content Screening, Staining