rabbit anti lamp2a pabs  (Proteintech)

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Analysis of Chaperone-Mediated Autophagy

doi: 10.1007/978-1-4939-8873-0_47

Figure Lengend Snippet: Chaperone-mediated autophagy: schematic model of the steps in chaperone-mediated autophagy.1. Substrate binding by HSC70 and cochaperones and targeting to lysosomes. 2. Binding of the substrate to LAMP2A at the lysosomal membrane. 3. HSP90 binds to LAMP2A to stabilize it while it organizes into higher molecular weight complexes. 4. Substrate crosses the lysosomal membrane through a LAMP2A-enriched translocation complex, and translocation is complete by the action of luminal HSC70. 5. The substrate is rapidly degraded by luminal proteases (cathepsins). 6. Once substrate translocation is complete, LAMP2A dissociates into monomers in a process dependent on cytosolic HSC70. Red box: negative regulators of CMA at the lysosomal membrane. Green box: positive regulators of CMA at the lysosomal membrane

Article Snippet: For hsc70, we recommend using IgM mouse monoclonal anti-hsc70 antibody clone 13D3 (available through several vendors) because most commercial antibodies recognize both hsp70 and hsc70, but clone 13D3 has been well characterized as specific for hsc70. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Type Source/cat# Dilution (IB) Dilution (IF) AKT Rabbit pAb IgG Cell signaling [9272] 1:1000 1: 200 pAKT (ser473) Rabbit pAb IgG Cell signaling [9271] 1:1000 1: 25 Cath A (A-19) Goat pAb IgG Santa Cruz [sc-26049] 1:500 N/A Cath D Goat pAb IgG Santa Cruz [sc-6486] 1:500 N/A EF1α Mouse mAb IgG Millipore [05–235] 1:1000 N/A GAPDH Rabbit mAb IgG Cell signaling [2118] 1:1000 1:100 GFAP Mouse mAb IgG Millipore [MAB360] 1:1000 1:200 pGFAP Rabbit pAb IgG ABGENT [AP3562a] 1:1000 N/A HSC70 (13D3) Mouse mAb IgM Novus biological [NB120–2788] 1:5000 1:500 HSP90 Rat mAb IgG ENZO 1:10,000 N/A LAMP-1 [H4A3] Mouse mAb IgG Abcam [ab25630] 1:3000 1:100 LAMP2A Rabbit pAb IgG ThermoFisher [51–2200] 1:1000 1:200 mTOR Rabbit pAb IgG Cell signaling [2972] 1:1000 N/A Rictor Rabbit mAb IgG Cell signaling [2114] 1:1000 N/A Rac1 Mouse mAb IgG Millipore 1:1000 N/A Ribonuclease A Rabbit pAb IgG Rockland immunochemicals 1:10,000 N/A Open in a separate window pAb polyclonal antibody, mAb monoclonal antibody Antibodies or CMA-related proteins and recommended dilutions Secondary antibodies: Fluorophores are selected depending on the combination of primary antibodies used, but common ones used in these procedures are Alexa Fluor488 goat-conjugated anti-mouse IgM antibody (ThermoFisher Scientific) (for antihsc70) and Alexa Fluro555 goat anti-rabbit IgG (ThermoFisher Scientific) (for anti-LAMP2A).

Techniques: Binding Assay, Molecular Weight, Translocation Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Analysis of Chaperone-Mediated Autophagy

doi: 10.1007/978-1-4939-8873-0_47

Figure Lengend Snippet: Antibodies or CMA-related proteins and recommended dilutions

Article Snippet: For hsc70, we recommend using IgM mouse monoclonal anti-hsc70 antibody clone 13D3 (available through several vendors) because most commercial antibodies recognize both hsp70 and hsc70, but clone 13D3 has been well characterized as specific for hsc70. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Type Source/cat# Dilution (IB) Dilution (IF) AKT Rabbit pAb IgG Cell signaling [9272] 1:1000 1: 200 pAKT (ser473) Rabbit pAb IgG Cell signaling [9271] 1:1000 1: 25 Cath A (A-19) Goat pAb IgG Santa Cruz [sc-26049] 1:500 N/A Cath D Goat pAb IgG Santa Cruz [sc-6486] 1:500 N/A EF1α Mouse mAb IgG Millipore [05–235] 1:1000 N/A GAPDH Rabbit mAb IgG Cell signaling [2118] 1:1000 1:100 GFAP Mouse mAb IgG Millipore [MAB360] 1:1000 1:200 pGFAP Rabbit pAb IgG ABGENT [AP3562a] 1:1000 N/A HSC70 (13D3) Mouse mAb IgM Novus biological [NB120–2788] 1:5000 1:500 HSP90 Rat mAb IgG ENZO 1:10,000 N/A LAMP-1 [H4A3] Mouse mAb IgG Abcam [ab25630] 1:3000 1:100 LAMP2A Rabbit pAb IgG ThermoFisher [51–2200] 1:1000 1:200 mTOR Rabbit pAb IgG Cell signaling [2972] 1:1000 N/A Rictor Rabbit mAb IgG Cell signaling [2114] 1:1000 N/A Rac1 Mouse mAb IgG Millipore 1:1000 N/A Ribonuclease A Rabbit pAb IgG Rockland immunochemicals 1:10,000 N/A Open in a separate window pAb polyclonal antibody, mAb monoclonal antibody Antibodies or CMA-related proteins and recommended dilutions Secondary antibodies: Fluorophores are selected depending on the combination of primary antibodies used, but common ones used in these procedures are Alexa Fluor488 goat-conjugated anti-mouse IgM antibody (ThermoFisher Scientific) (for antihsc70) and Alexa Fluro555 goat anti-rabbit IgG (ThermoFisher Scientific) (for anti-LAMP2A).

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Analysis of Chaperone-Mediated Autophagy

doi: 10.1007/978-1-4939-8873-0_47

Figure Lengend Snippet: Measurement of CMA in cultured cells and in vivo. Top: functional assays, expression of photoswitchable reporters (left) allows quantification of CMA in cultured cells as the number of fluorescent puncta (lysosomes) per cell. Injection of leupeptin in vivo (to block lysosomal proteolysis) and immunoblot for CMA substrate of lysosomes from injected and not injected mice allows quantification of substrate flux by CMA (right). Bottom: steady-state assays, quantification of number of lysosomes positive for LAMP2A and HSC70 allows for detecting changes in lysosomes capable of performing CMA (left). Immunoblot of isolated lysosomes for well-characterized CMA activator and inhibitor proteins. Profile of differences in specific protein levels in lysosomes with upregulated and downregulated CMA activity are shown

Article Snippet: For hsc70, we recommend using IgM mouse monoclonal anti-hsc70 antibody clone 13D3 (available through several vendors) because most commercial antibodies recognize both hsp70 and hsc70, but clone 13D3 has been well characterized as specific for hsc70. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Type Source/cat# Dilution (IB) Dilution (IF) AKT Rabbit pAb IgG Cell signaling [9272] 1:1000 1: 200 pAKT (ser473) Rabbit pAb IgG Cell signaling [9271] 1:1000 1: 25 Cath A (A-19) Goat pAb IgG Santa Cruz [sc-26049] 1:500 N/A Cath D Goat pAb IgG Santa Cruz [sc-6486] 1:500 N/A EF1α Mouse mAb IgG Millipore [05–235] 1:1000 N/A GAPDH Rabbit mAb IgG Cell signaling [2118] 1:1000 1:100 GFAP Mouse mAb IgG Millipore [MAB360] 1:1000 1:200 pGFAP Rabbit pAb IgG ABGENT [AP3562a] 1:1000 N/A HSC70 (13D3) Mouse mAb IgM Novus biological [NB120–2788] 1:5000 1:500 HSP90 Rat mAb IgG ENZO 1:10,000 N/A LAMP-1 [H4A3] Mouse mAb IgG Abcam [ab25630] 1:3000 1:100 LAMP2A Rabbit pAb IgG ThermoFisher [51–2200] 1:1000 1:200 mTOR Rabbit pAb IgG Cell signaling [2972] 1:1000 N/A Rictor Rabbit mAb IgG Cell signaling [2114] 1:1000 N/A Rac1 Mouse mAb IgG Millipore 1:1000 N/A Ribonuclease A Rabbit pAb IgG Rockland immunochemicals 1:10,000 N/A Open in a separate window pAb polyclonal antibody, mAb monoclonal antibody Antibodies or CMA-related proteins and recommended dilutions Secondary antibodies: Fluorophores are selected depending on the combination of primary antibodies used, but common ones used in these procedures are Alexa Fluor488 goat-conjugated anti-mouse IgM antibody (ThermoFisher Scientific) (for antihsc70) and Alexa Fluro555 goat anti-rabbit IgG (ThermoFisher Scientific) (for anti-LAMP2A).

Techniques: Cell Culture, In Vivo, Functional Assay, Expressing, Injection, Blocking Assay, Western Blot, Isolation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: Neither chaperone-mediated autophagy nor Nedd4/Ndfip-1 accounts for the SB203580 effect to increase αSyn turnover. A – D , NGF-differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing scrambled or LAMP2a shRNA, and after 2 days, lysates were analyzed for LAMP2a and actin, and the medium for secreted αSyn and LDH. B , quantitation of LAMP2a shRNA knockdown effect (one-way ANOVA, N = 5). C , conditioned medium from PC12-αSyn/p25α cells expressing scrambled or LAMP2a shRNA and treated with/without SB203580 was analyzed for αSyn by Western blotting (one-way ANOVA, N = 5). D , LDH content in conditioned medium (one-way ANOVA, N = 5). E , indirect immunofluorescence of αSyn (mAb LB509; in green ) and LAMP2a (in red ) in PC12-αSyn/p25α cells treated with 1 μM SB203580 and 10 μM E64 to inhibit lysosomal proteases. A representative image after analysis in ImageJ with a colocalization algorithm is shown, where colocalized pixels (if present) appear white . Note the peripheral localization of αSyn-positive vesicles, mostly separated from the perinuclear LAMP2a-positive lysosome pool ( inset ; pixel intensity scatter plot) yielding a Pearson coefficient of −0.08 ± 0.01 (n = 11 cells from two experiments). Bar represents 10 μm. F , conditioned medium from PC12-αSyn/p25α cells treated with/without SB203580 was either differentially centrifuged to obtain a washed exosomal pellet (Exo) or TCA-precipitated (TCA), and fractions were then analyzed by Western blotting for αSyn and Ndfip1. Note the presence of αSyn monomer (m) and oligomers (oli) in both exosome and concentrated supernatant (TCA) fractions, and that SB203580 increases Ndfip1 in exosomes, while decreasing αSyn. The shown blot is representative of two independent trials; all lanes are derived from the same membrane for αSyn and Ndfip1, respectively. Molecular weight markers are indicated. G , indirect immunofluorescence to show αSyn (mAb LB509) in relation to Ndfip1 and LAMP1 in PC12 cells expressing αSyn/p25α under basal conditions. Arrows indicate colocalization of αSyn, Ndfip1, and LAMP1. The shown images are representative of two independent experiments. The squared area in top right panel is depicted at higher magnification in the lower row of panels . Bar upper panels represent 10 μm; bar lower panels represent 2.5 μm. H , a representative image after analysis in the ImageJ colocalization algorithm is shown, where αSyn ( red ) and Ndfip1 ( green ) pixels appear white when colocalized ( arrows ). Analysis of αSyn/Ndfip1 colocalization yielded a Pearson coefficient of 0.13 ± 0.02 (n = 10 cells from a single representative experiment of two). Bar represents 10 μm. I – K , NGF-differentiated PC12-αSyn/p25α cells were transduced with lentivectors expressing scrambled or Ndfip1 shRNA and then treated with doxycycline for 2 days with/without SB203580 as indicated. I , representative Western blots are shown for Ndfip1 protein in the lysate and for secreted αSyn in the conditioned medium. J , quantitation of Ndfip1 knockdown (one-sample t test, N = 4). K , quantitation of αSyn release from PC12-αSyn/p25α cells that received scrambled or Ndfip1-shRNA treated or not with 1 or 2 μM SB203580 as indicated (one-way ANOVA, N = 4). All graphs show mean ± SEM. αSyn, α-synuclein; LAMP, lysosome-associated membrane protein; LDH, lactate dehydrogenase; mAb, monoclonal antibody; NGF, nerve growth factor; TCA, trichloroacetic acid.

Article Snippet: Antibodies used included mouse total anti-αSyn monoclonal antibodies (mAbs) (catalog no.: 610787; BD Transduction Laboratories), 4B12 (catalog no.: MA1-90346; Invitrogen), LB509 (catalog no.: sc-58480; Santa Cruz), p-Ser129 αSyn (catalog no.: AB51253; Abcam), and rabbit anti-αSyn pAb (catalog no.: S3062; Sigma); rabbit anti-LC3B (catalog no.: L7543; Sigma or catalog no.: NB600-1364, Novus); anti-p62/SQSTM1 (catalog no.: P0067, Sigma; catalog no.: 51145, Cell Signaling); rabbit anti-LAMP2A pAb (catalog no.: ab18528, Abcam); rabbit anti-LAMP1 pAb (kind gift of Dr Sven Carlsson, Umeå University, Sweden); rat anti-hsc70 mAb (catalog no.: Ab19136, Abcam); rabbit anti-p38MAPKα (catalog no.: 9218, Cell Signaling Technology in A ; catalog no.: SMC-152D from StressMarq Biosciences in G ), and anti-p38MAPKγ (catalog no.: 2307, Cell Signaling) pAbs; p-p38MAPK was detected by mAb (catalog no.: 4511S, Cell Signaling Technology), and total p38MAPK was detected by rabbit pAb (catalog no.: 9212; Cell Signaling Technology); anti-mouse β-actin (catalog no.: A5441, Sigma), Map2 (catalog no.: M4403, Sigma), and ubiquitin (catalog no.: VU1, Synaptic Systems) mAbs were used.

Techniques: Transduction, Expressing, shRNA, Quantitation Assay, Knockdown, Western Blot, Immunofluorescence, Derivative Assay, Membrane, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: α-synuclein buildup is alleviated via ESCRT-dependent endosomal degradation brought about by p38MAPK inhibition in cells expressing p25α

doi: 10.1016/j.jbc.2022.102531

Figure Lengend Snippet: αSyn degradation under basal conditions and p38MAPK inhibition. Under basal conditions αSyn is degraded by multiple mechanisms first and foremost by macroautophagy and Ndfip1-mediated internalization of αSyn; however, chaperone-mediated autophagy (LAMP2a and Hsc70) also contributes. When p25α-mediated p38MAPK activation is opposed pharmacologically or genetically, the majority of αSyn is turned over in an ESCRT-dependent process relying on Hsc70 and TSG101, and αSyn degradation commences in late endosomes. Under basal conditions, lysosome fusion with autophagosomes and amphisomes (the fusion product of an autophagosome with a late endosome) is partially blocked by p25α, which results in their exocytosis and release of αSyn. In contrast, under conditions of p38MAPK inhibition, ESCRT-dependent αSyn import and degradation begins in the late endosome, and the endosomal pathway runs to completion to deliver αSyn to lysosomes for degradation. αSyn, α-synuclein; ESCRT, endosomal sorting complex required for transport; LAMP, lysosome-associated membrane protein.

Article Snippet: Antibodies used included mouse total anti-αSyn monoclonal antibodies (mAbs) (catalog no.: 610787; BD Transduction Laboratories), 4B12 (catalog no.: MA1-90346; Invitrogen), LB509 (catalog no.: sc-58480; Santa Cruz), p-Ser129 αSyn (catalog no.: AB51253; Abcam), and rabbit anti-αSyn pAb (catalog no.: S3062; Sigma); rabbit anti-LC3B (catalog no.: L7543; Sigma or catalog no.: NB600-1364, Novus); anti-p62/SQSTM1 (catalog no.: P0067, Sigma; catalog no.: 51145, Cell Signaling); rabbit anti-LAMP2A pAb (catalog no.: ab18528, Abcam); rabbit anti-LAMP1 pAb (kind gift of Dr Sven Carlsson, Umeå University, Sweden); rat anti-hsc70 mAb (catalog no.: Ab19136, Abcam); rabbit anti-p38MAPKα (catalog no.: 9218, Cell Signaling Technology in A ; catalog no.: SMC-152D from StressMarq Biosciences in G ), and anti-p38MAPKγ (catalog no.: 2307, Cell Signaling) pAbs; p-p38MAPK was detected by mAb (catalog no.: 4511S, Cell Signaling Technology), and total p38MAPK was detected by rabbit pAb (catalog no.: 9212; Cell Signaling Technology); anti-mouse β-actin (catalog no.: A5441, Sigma), Map2 (catalog no.: M4403, Sigma), and ubiquitin (catalog no.: VU1, Synaptic Systems) mAbs were used.

Techniques: Inhibition, Activation Assay, Membrane