Rabbit anti lamin b1

Nuclear proteins were extracted from hypoxic and normoxic cells using a NE-PER nuclear and cytoplasmic extraction kit (Cat# 78833, Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of nuclear protein were electrophoresed in 10% SDS-polyacrylamide gels and transferred to hybond-PVDF membranes (Cat# 23225, GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used in this study were: rabbit anti-Lamin B1 (Cat# ab133741, Abcam, Cambridge, MA, USA) and rabbit anti-HIF-1α (D2U3T) (Cat# 14179, Cell Signaling Technology, Danvers, MA, USA). Next, the membranes were incubated with anti-rabbit Ig, peroxidase-linked, species-specific whole antibody (Cat# 31460, Thermo-Fisher Scientific). ECL reagents were used to detect the signals according to the manufacturer’s instructions (Cat# RPN3244, GE Healthcare Life Sciences, Pittsburgh, PA, USA). All films were scanned with an Epson Expression 1680 optical scanner (Epson America Inc., Long Beach, CA, USA). Densitometry analysis of bands was performed using an open-source, public domain software package (ImageJ v1.47).

Non-neurological control and C9orf72 patient postmortem paraffin embedded motor cortex sections were obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 5: Supplemental Table 2 for demographic information). Antigen retrieval and immunofluorescent staining was conducted as previously described [3 (link)]. Antibodies for immunostaining are as follows: 1:500 Rabbit Anti-Lamin B1 (Abcam ab16048), 1:1000 Guinea Pig Anti-Map2 (Synaptic Systems 188,004), 1:1000 Goat Anti-Guinea Pig Alexa 488 (Invitrogen A11073), 1:1000 Goat Anti-Rabbit Alexa 647 (Invitrogen A21245). Nuclei from Map2 positive Layer V neurons were imaged with a 63X objective and a Zeiss Axioimager Z2 fluorescent microscope equipped with an apotome2 module. All images were acquired using identical exposure times. Images are presented as default apotome processed images generated in Zeiss Zen Blue 2.3.

Whole cell lysates were prepared using SDS-PAGE sample buffer supplemented with benzonase nuclease (Sigma, E1014) and boiled for 5 min. Proteins were separated on SDS-PAGE gels (4–20% gradient) and transferred to PVDF membrane. Membranes were blocked in Odyssey PBS Blocking Buffer (Li-Cor, 927–40,000). The primary antibodies used were mouse anti-NTF2 at 1:500 (Sigma, N9527), rabbit anti-NTF2 at 1:200 (Aviva System Biology, ARP64840_P050), mouse anti-lamin A/C at 1:500 (Santa Cruz Biotech sc-376248), rabbit anti-lamin B1 at 1:2000 (Abcam 16,048), and mouse anti-actin at 1:200 (Abgent, AM1965b). The secondary antibodies were IRDye 680RD anti-mouse used at 1:20,000 (Li-Cor 925-68070) and IRDye 800CW anti-rabbit used at 1:20,000 (Li-Cor 925-32211). Blots were scanned on a Li-Cor Odyssey CLx instrument and band quantification was performed with ImageStudio. For a given sample, NTF2 band intensity was normalized to the actin signal, and lamin band intensity was normalized to Ponceau-stained total protein. For western blots on LNCaP cell lysates, HRP detection was used as previously described^{73 (link)}.

Proteins extracted from microglia, astrocytes and brain tissues (after perfusion as described in Sect. Imaging of labeled platelets) were processed and subjected to immunoblotting using the Simple Wes System (Protein Simple, USA) and Size Separation Master Kit with Split Buffer (12–230 kDa) according to the manufacturer’s instructions. The following primary antibodies were used: rabbit anti-nuclear factor kappa B inhibitor alpha (1:30; IκBα; Abcam), rabbit anti-nuclear factor kappa B P65 (1:10; P65-NFκB; Abcam), rabbit anti-pP65-NFκB (1:20; Invitrogen), rabbit anti-CD41 (1:20; Abcam), mouse anti-β-actin (1:50; Abcam), and rabbit anti-Lamin B1 (1:50; Abcam). The immunoblots were analyzed using Compass software 4.0.0 (Protein Simple).

To evaluate CHOP expression, nuclear and cytosolic fractions were prepared from BMM using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Proteinase inhibitor cocktail tablets (EDTA-free) (Roche) were added to the buffers. For immunoblotting, cytoplasmic and nuclear fractions were diluted with 5× sample buffer (Thermo Fisher Scientific) and 10× reducing agent (Thermo Fisher Scientific), then incubated in boiling water for 5 min. Proteins were separated on 4–20% Tris/Glycine gels (BioRad) and immunoblotting was performed as described^{32 (link)}. The following primary antibodies were used: mouse anti-CHOP (Cell Signaling Technology, #2895), rabbit anti-Lamin B1 (Abcam, #ab16048), and mouse anti-GAPDH (Santa Cruz Biotechnologies, #sc-32233). HRP-conjugated secondary antibodies (R&D Systems, #HAF008, #HAF007) against their respective primary antibodies were incubated with membranes for 1 h at room temperature. Proteins were visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).