anti β actin antibody rhodamine  (Bio-Rad)

Journal: bioRxiv

Article Title: Missense variants in GABA A receptor beta2 subunit disrupt receptor biogenesis and cause loss of function

doi: 10.1101/2025.03.09.642292

Figure Lengend Snippet: β2 missense epilepsy-associated variants (EAVs) in different region of the subunit lead to differing total protein expression and increased tendency to aggregate. (A) Cartoon representation of the pentameric GABA A receptor (GABA A R), consisting of α-β-α-β-γ subunit assembled in clockwise direction when viewed from the synaptic cleft. PDB: 6X3S. (B) Structure of GABA A R, highlighting the positions of selected four missense variants in β2 subunit. Q209F210delinsH and R240T are located in the N-terminal domain near the entrance of the first transmembrane (TM) helix; I246T situates in the beginning of TM1; and I299S is in the extracellular TM2-3 loop region. (C) HEK293T cells transiently expressing β2 missense variants with WT α1 and WT γ2 (1:1:1; 0.25 µg each) were harvested forty-eight hours post transfection. Cells were lysed and solubilized in 2 mM n-dodecyl-β-maltoside (DDM). Total proteins were then subjected to SDS-PAGE and western blot to visualize the expression of α1, β2 and γ2 subunits. β2 protein displayed two bands at 55 and 48 kDa, respectively, which represented distinct glycoforms. β-actin was used as a loading control. Band intensity was quantified with ImageJ and normalized to the loading control, which is shown on the right (n=4). (D) Quantification of individual β2 glycoform band and the ratio of upper to lower band from (C). (E) Aggregation propensity of β2 EAVs. Cell pellet after extraction of soluble proteins (i.e., detergent-insoluble fraction) was washed and resuspended in Laemmli sample buffer containing 2% SDS and proceeded with western blot analysis. Band intensity of the entire lane was quantified, as large aggregates are seen at molecular weight above 250 kD. Insoluble-to-soluble-β2 ratio was calculated as a measure of aggregation propensity, which is shown on the right (n =4). Data is presented as mean ± SEM. One-way ANOVA followed by a post-hoc Dunnett’s test was used to determine statistical significance. * p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The following primary antibodies were used: the mouse monoclonal anti-GABA A α1 subunit antibody (1:2000; catalog #: MAB339), mouse monoclonal anti-GABA A β2/3 subunit antibody (1:1000, catalog #: 05–474), rabbit poly-clonal anti-GABA A β2 subunit antibody (1:1000, catalog #: AB5561), rabbit polyclonal anti-GABA A γ2 subunit antibody (1:1000, catalog #: AB5559), rabbit monoclonal anti-Na + /K + ATPase antibody (1:10,000; catalog #: ab76020), mouse monoclonal anti-ubiquitin antibody (1:2000; catalog #: 14-6078-82), rabbit polyclonal anti-LC3b antibody (1:2000; catalog #: NB100-2220), and the fluorescent hFAB Rhodamine anti-β-actin antibody from Biorad (catalog #: 12004163).

Techniques: Expressing, Transfection, SDS Page, Western Blot, Control, Extraction, Molecular Weight

Journal: bioRxiv

Article Title: Missense variants in GABA A receptor beta2 subunit disrupt receptor biogenesis and cause loss of function

doi: 10.1101/2025.03.09.642292

Figure Lengend Snippet: β 2 EAVs engage both proteasomal and lysosomal degradation pathways. ( A ) To evaluate the degree of proteasomal and lysosomal degradation, HEK293T cells transiently expressing β2 EAVs were treated with MG132 (10 µM, 6 hrs) or bafilomycin A1 (bafA1, 1 µM, 6 hrs) to inhibit the proteasome or lysosome, respectively. Cells were lysed forty-eight after transfection and total protein was visualized using SDS-PAGE and western blot analysis (n=4). ( B ) Quantification of band intensity normalized to the loading control (β-actin). ( C ) Ubiquitination levels of β2 EAVs using a co-immunoprecipitation (co-IP) assay. HEK293T cells were co-transfected with HA-tagged ubiquitin along with WT or β2 EAVs. Cells were treated with MG132 (10 µM, 6 hrs) to inhibit proteasomal degradation and harvested forty-eight hours post transfection. 1000 µg of total lysates were pre-cleared and incubated with anti-GABA A R β2 antibody, followed by incubation with protein A/G beads to pull down β2 and β2-interacting proteins. Eluent from the beads were proceeded with SDS-PAGE and western blot to detect ubiquitin levels. Quantification of Ubiquitin, which is normalized to the immunoprecipitated β2, post co-IP is shown on the right (n=3). IP: immunoprecipitation. Data is presented as mean ± SEM. One-way ANOVA followed by a post-hoc Dunnett’s test was used to determine statistical significance. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: The following primary antibodies were used: the mouse monoclonal anti-GABA A α1 subunit antibody (1:2000; catalog #: MAB339), mouse monoclonal anti-GABA A β2/3 subunit antibody (1:1000, catalog #: 05–474), rabbit poly-clonal anti-GABA A β2 subunit antibody (1:1000, catalog #: AB5561), rabbit polyclonal anti-GABA A γ2 subunit antibody (1:1000, catalog #: AB5559), rabbit monoclonal anti-Na + /K + ATPase antibody (1:10,000; catalog #: ab76020), mouse monoclonal anti-ubiquitin antibody (1:2000; catalog #: 14-6078-82), rabbit polyclonal anti-LC3b antibody (1:2000; catalog #: NB100-2220), and the fluorescent hFAB Rhodamine anti-β-actin antibody from Biorad (catalog #: 12004163).

Techniques: Expressing, Transfection, SDS Page, Western Blot, Control, Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation

Journal: eLife

Article Title: Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum

doi: 10.7554/eLife.84798

Figure Lengend Snippet:

Article Snippet: Antibody , Fluorescent anti-β-actin Rhodamine , Biorad , Cat#: 12004163 , WB (1:10,000).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Cloning, Software